"Ro-Kyu-Bu!" centers around Subaru Hasegawa, a high school student and former basketball player who is forced to quit his team due to a scandal involving the coach. When his aunt, Mihoshi Takamura, asks him to coach her elementary school's girls' basketball team, Subaru reluctantly agrees. The team, Tomoka Minato, Maho Misawa, Airi Kashii, Saki Nagatsuka, and Hinata Hakamada, initially struggles with their skills and teamwork but gradually improves under Subaru's guidance. The narrative explores their growth as players and individuals, balancing lighthearted moments with the challenges of competitive sports.

The series delves into the dynamics between Subaru and the girls, highlighting their determination to overcome obstacles and prove themselves in tournaments. Subaru's coaching methods focus on fostering teamwork and confidence, while the girls' personalities and backstories add depth to their interactions. Tomoka, the team's ace, is passionate but struggles with self-doubt, while Maho, the captain, provides a steadying presence. Airi, Saki, and Hinata each bring unique strengths and quirks to the team, creating a cohesive yet diverse group.

As the story progresses, the team faces rival schools and personal challenges, culminating in their participation in a regional tournament. The series emphasizes themes of perseverance, friendship, and the joy of sports, blending comedic and heartfelt moments. While the focus remains on basketball, the relationships between the characters and their individual growth are integral to the narrative. "Ro-Kyu-Bu!" balances its sports-centric plot with slice-of-life elements, offering a mix of competition and character-driven storytelling.