Abstract: With the advancement of human space exploration, the effects of the space environment on human cells have attracted increasing attention from scientists and constitute an important component of space biology. This review focuses on the impact of microgravity on tumor cells and its potential applications in cancer therapy. It discusses in detail how microgravity influences tumor cell proliferation, apoptosis, migration, immune response, and alterations in tumor cell stemness through the regulation of relevant molecules and signaling pathways. The review also describes the advantage of microgravity in promoting the spontaneous formation of three-dimensional cell aggregates, which facilitates anticancer drug screening and research on tumor drug resistance. Furthermore, it explores the effects of microgravity on the efficacy of anticancer therapeutics and compares real microgravity in space with ground-based simulated microgravity systems. In summary, the effects of microgravity on tumor cells are multifaceted, and achieving a comprehensive understanding and targeted modulation of these effects represents a critical direction for future research efforts.