"Tezuka Osamu ga Kieta?! 20 Seiki Saigo no Kaijiken" is a two-episode anime special that blends mystery, science fiction, and historical elements. The narrative revolves around the sudden disappearance of Osamu Tezuka, a legendary manga artist, in 1989. His vanishing sparks a nationwide investigation led by Detective Shunsaku Ban, a character originally created by Tezuka himself. Ban is joined by other iconic Tezuka characters, including Rock Holmes and Mustachio, as they delve into the mystery.

The investigation uncovers a series of bizarre events tied to a secretive organization known as the "Black Shadow." This group is revealed to be manipulating time and space, using advanced technology to alter historical events. The story takes a surreal turn as the characters travel through different time periods, encountering historical figures and witnessing pivotal moments in 20th-century history. The narrative explores themes of creativity, legacy, and the impact of art on society, as Tezuka's works and characters play a central role in unraveling the mystery.

The plot thickens when it is revealed that Tezuka's disappearance is linked to a larger conspiracy aimed at erasing his influence from history. The Black Shadow seeks to eliminate Tezuka's contributions to manga and animation, believing his work has the power to inspire change and challenge their control. The characters must navigate a labyrinth of time loops, alternate realities, and existential threats to rescue Tezuka and restore the timeline.

The anime features a rich ensemble of Tezuka's beloved characters, each contributing their unique skills and personalities to the investigation. Detective Ban serves as the logical and determined leader, while Rock Holmes provides youthful energy and resourcefulness. Mustachio adds a comedic yet brave presence, balancing the tension with lighthearted moments. The interplay between these characters drives the narrative forward, blending action, humor, and emotional depth.

As the story progresses, the stakes escalate, culminating in a climactic confrontation with the Black Shadow. The resolution emphasizes the enduring power of creativity and the importance of preserving cultural heritage. The anime pays homage to Tezuka's legacy by weaving his creations into a meta-narrative that celebrates his impact on the medium while delivering an engaging and thought-provoking mystery.