In the spring of 2026, a special crossover event brings together two generations of horse racing anime. A small, odd-looking white mule named Midori Makibao, who dreams of becoming a champion racehorse despite his unusual appearance, finds himself transported into the vibrant world of Umamusume: Pretty Derby. This world is inhabited by horse girls, young women who possess the ears and tails of horses and inherit the names and spirits of legendary racehorses, training at the prestigious Tracen Academy to compete in the Twinkle Series.

The unlikely meeting occurs as part of a celebration marking the 30th anniversary of the Midori no Makibao manga and the 5th anniversary of the Umamusume game, set during the Year of the Horse. Makibao, a character known for his gritty determination and comedic stubbornness, is voiced by his original voice actress Inuko Inuyama, maintaining his iconic personality. He is immediately confronted by several prominent horse girls, including the energetic and talented Tokai Teio, the graceful and prideful Mejiro McQueen, the speedy Sakura Bakushin O, the powerful TM Opera O, and the experienced Sakura Chiyono O. These girls are accustomed to competing on equal footing, but the arrival of a real, talking horse presents an unprecedented challenge.

The central conflict arises from a fundamental difference in racing philosophy and physical form. Makibao races on all fours, relying on raw guts, a unique running style called the Mustang Special, and an unorthodox training regimen taught by his rat companion Chuubei. In contrast, the horse girls race on two legs, combining athleticism with idol-like performances and sophisticated strategies. An exhibition race is arranged to settle the question of who is the faster runner. As Makibao struggles to adapt to the rules and social norms of Tracen Academy, he teaches the horse girls a lesson about pure, unbreakable spirit and the harsh realities of the racetrack, while they help him understand the importance of teamwork and the joy of running for a crowd that cheers his name.

Notable narrative arcs within this collaboration include the Crossover Video arc, titled Run OO O, where Makibao appears alongside horse girls whose names all end with the letter O, showcasing a series of playful interactions and a relay race set to a new arrangement of the classic Midori no Makibao opening theme Hashiro Makibao. Another arc focuses on the Training Camp Clash, where Makibao refuses to follow standard training protocols, leading to a comedic yet intense series of workouts that push both him and his horse girl rivals to their limits. The story ultimately builds to the Legend vs. Legacy Race, a special event where the outcome is less about victory and more about mutual respect, highlighting how the fighting spirit of underdogs from the past can inspire a new generation of champions. The collaboration serves as a nostalgic tribute and a passing of the torch, celebrating the enduring appeal of horse racing stories across different eras.