Soren, a young Barn owl, lives in the Tyto Forest with his family: his father, Noctus; his mother, Marella; his older brother, Kludd; his younger sister, Eglantine; and Mrs. P, the family’s nest maid, a kindly blind snake. Soren enjoys listening to the ‘Legends of Ga’Hoole,’ which are mythical tales of warrior owls called the Guardians who fight against the Pure Ones. Kludd berates his brother’s dreams, having grown tired of listening to the stories. When their father teaches them the first steps to flying, Kludd becomes jealous of Soren’s natural branching ability. Later, when practicing branching while their parents are hunting, Kludd pushes Soren off a branch which forces them both to fall to the ground. They are attacked by a Tasmanian devil but are saved, and then kidnapped, by two owls, Jatt and Jutt.
Along the way, more owlets are revealed to have been kidnapped. Soren meets Gylfie, a young Elf owl who is also kidnapped. At St. Aegolius, the owlets are greeted by Nyra, who says that they will be divided into either soldiers or ‘pickers’. When Soren and Gylfie protest, they are sorted as pickers while Kludd, who betrays Soren, is sent to be a soldier. Soren and Gylfie avoid being ‘moon-blinked’ (a catatonic state induced by sleeping under a full moon), while the others do not know and become moon-blinked. They are forced to ‘pick’ through owl’s pellets, scouring for bits of metal ‘flecks’, which are being used to build a superweapon. They are spotted by Grimble, a Boreal owl, who reveals that he works under the Pure Ones only to keep his family safe and that he isn’t really a Pure One at all. He teaches them to fly, but Nyra finds out whilst coming to invite Soren to become a soldier. Kludd decides to stay behind and become a Pure One, despite Soren’s pleas for him to come with him. Grimble and Nyra engage in a battle, but Grimble is eventually killed. Following his instructions to seek the Great Tree of Ga’Hoole and warn the Guardians, Soren and Gylfie fly out. Along their journey, they meet Twilight, a Great grey owl, and Digger, a Burrowing owl, and rest with them in their shared hollow. Soren is reunited with Mrs. P who is brought in by Twilight, having captured her for dinner while she searched for them. She agrees to go with them to find the Guardians.
The band flies towards the sea of Hoolemere where a flock of crows chaotically guides them to the legendary shrine and gateway of the Guardians, kept by an oracular echidna, who provides comic, but accurate, descriptions of the quintet and guidance to the object of their quest. He sends the owls out to sea to find the island of Ga’Hoole. En route, the band encounters a fierce storm and Digger’s wings freeze, causing him to nearly drown in the sea, but they are saved by Boron and Barran, the king and queen of the Guardians who lead them back to the Tree of Ga’Hoole. There, Soren tells the Guardian council about the Pure One’s plans. The Guardians are skeptical, but an elderly Screech owl named Ezylryb is convinced by Soren’s sincerity. Boron sends a Great grey owl called Allomere out with two scouts to investigate St. Aegolius.
The band is shown refuge at the Tree of Ga’Hoole, guided by a young Guardian-in-training named Otulissa, a Short-eared owl. Soren develops a crush on her, to Gylfie’s annoyance. They are sent to attend different classes called ‘chaws’. During one lesson in the middle of a rainstorm, Soren grasps a brief hold on gizzard-flying (flying purely with instinct), but loses control and falls towards the sea. Ezylryb rescues him and orders Soren back to his hollow. There, Soren learns that Ezylryb is, in fact, the Lyze of Kiel; the legendary leader of the Guardians who fought and defeated Metal Beak, the ancient leader of the Pure Ones, and Nyra’s mate. But Soren is disheartened when he learns that his hero is a battle worn veteran who scoffs at his own past. Allomere returns from his scouting mission with news that his two wingmen were killed in an ambush. He brings back with him two moon-blinked owlets, one of which is Soren’s sister, Eglantine. Seeing the evidence, the Guardians arrange for battle and fly out towards St. Aegolius. Before leaving, Ezylryb instructs an anxious Soren to tend to his sister. When she finally snaps out of her trance, Eglantine tells Soren that it was Kludd who moon-blinked her and gave her to Allomere, and he realizes that Allomere is a traitor and that the Guardians are actually flying into a trap. The band then follows the Guardians where they find the Guardians subdued by the flecks, contained in a machine operated by bats.
Twilight, Gylfie and Digger fend off the bats sent by Metal Beak to kill the paralyzed Guardians. This causes Metal Beak to distrust Allomere, and, believing he lied to him about bringing all the Guardians, has him dragged away by several bats, and is seemly killed in the darkness. Meanwhile, Soren flies through a forest fire, igniting a lamp of oil, and manages to disable the fleck trap. Freed and recovered, the Guardians fly into battle and Ezylryb goes straight for Metal Beak, who with Nyra begins to overpower him. Soren flies into the battle after spotting Kludd, but Kludd attacks him, throwing him into the forest fire. The two brothers briefly fight while Soren tries to talk some sense into Kludd but Kludd replies that the Pure Ones believe in him like no one ever has and how Metal Beak said that the strong with triumph, the broken will be put out of their misery and that honor is just another word for weakness. Soren replies that he knows that Kludd doesn’t think like that to which Kludd replies that Soren doesn’t know him at all, before he leaps onto Soren, causing them to fall down a tree. Injured, Kludd tries to trick Soren to help him up, but as he attempts to do so, Kludd attacks him again, only for the branch Kludd is holding onto to snap, causing him to fall into the fire and disappear, seemingly killed. Furious at Kludd’s apparent death and betrayal, Soren grabs a flaming branch and attacks Metal Beak. Metal Beak easily has the upper hand on Soren, but grows over-confident and Soren manages to kill him, impaling him with the branch when he dives at Soren. Nyra, shocked at her mate’s death, retreats with the remaining Pure Ones.
When they return to the tree, Soren and Eglantine are reunited with their parents, who have come to the tree. The band is then welcomed as new Guardians. Soren states that whilst Metal Beak was defeated, Nyra escaped and Kludd’s body was never found. Meanwhile, back in the smoking remains of the canyons, a shadowy owl; presumably Kludd, is shown staring at the mask of Metal Beak with red “Pure One” eyes.