"My Friend's Little Sister Has It In for Me!" centers around Akiteru Daiki, a high school student and aspiring light novel author. He collaborates with his childhood friend, Otoi Nanami, who illustrates his stories under the pen name "Kageishi Iroha." Together, they form a successful creative duo, but their partnership is complicated by the arrival of Otoi's younger sister, Mashiro Nanami. Mashiro, a popular and talented actress, harbors a deep grudge against Akiteru, believing he has stolen her sister's attention and affection.

Mashiro enrolls in the same school as Akiteru and Otoi, determined to disrupt their relationship and sabotage Akiteru's career. Despite her antagonistic behavior, Mashiro's feelings are more complex than they initially appear, as her actions often reveal a mix of jealousy, insecurity, and a desire for validation. Akiteru, meanwhile, struggles to navigate Mashiro's schemes while maintaining his creative partnership with Otoi and pursuing his dream of becoming a successful author.

The story explores themes of rivalry, ambition, and the complexities of relationships, blending comedy, drama, and light romantic tension. Akiteru's interactions with Mashiro and Otoi are central to the narrative, as the trio's dynamic shifts between collaboration, conflict, and moments of genuine connection. The series also delves into the challenges of creative work, portraying the pressures and rewards of writing and illustrating in the competitive world of light novels. Through its engaging characters and layered interpersonal dynamics, the manga offers a nuanced portrayal of ambition, rivalry, and the bonds that connect people.