"Ansatsu Kyōshitsu" is set in a world where a powerful, octopus-like creature, Koro-sensei, has destroyed 70% of the Moon, rendering it permanently crescent-shaped. Koro-sensei announces his intention to destroy Earth within a year but offers humanity a chance to stop him by becoming the homeroom teacher of Class 3-E at Kunugigaoka Junior High School. This class, composed of students deemed academic failures, is tasked with assassinating him before the deadline. The Japanese government offers a substantial monetary reward to the student who succeeds, while the students themselves are motivated by the opportunity to improve their skills and prove their worth.

The narrative centers on the students of Class 3-E as they balance their academic responsibilities with their assassination training. Koro-sensei, despite his destructive potential, proves to be an exceptional teacher, nurturing the students' individual talents and fostering their personal growth. His methods are unconventional, often blending humor and life lessons with combat training. The students, initially seen as outcasts, gradually develop confidence and camaraderie as they work together to devise creative strategies to eliminate their target.

Key characters include Nagisa Shiota, a quiet and observant student who excels in assassination techniques due to his natural stealth and analytical mind. Karma Akabane, a rebellious and highly skilled student, serves as a foil to Nagisa, often taking a more aggressive approach. Other notable students include Kaede Kayano, who harbors a mysterious connection to Koro-sensei, and Rio Nakamura, a pragmatic and resourceful class representative. The class is also supported by their human teacher, Irina Jelavić, a professional assassin posing as an English instructor, and Tadaomi Karasuma, a government agent who trains the students in combat.

As the story progresses, the students uncover Koro-sensei's tragic backstory, revealing his origins and the reasons behind his destructive actions. This revelation adds depth to the narrative, as the students grapple with their conflicting emotions about their mission. The series explores themes of redemption, the value of education, and the importance of empathy, all while maintaining a balance of action, humor, and emotional depth. The students' journey culminates in a climactic confrontation that tests their resolve and the bonds they have formed, ultimately leading to a resolution that ties together the series' central themes and character arcs.