OBJECTIVES: Human minor salivary gland stem cells (huMSGSCs) are promising in regenerative medicine. Their multipotent capabilities enable tissue regeneration and offer treatment potential for various diseases. The effects of hypergravity (HyperG) and microgravity (MicroG) on stemness and therapeutic potential are not well explored. Therefore, this study investigated the effects of short-term HyperG and MicroG exposure on huMSGSC stemness and differentiation potential for treating salivary gland dysfunction. METHODS: huMSGSCs were exposed to 1G, MicroG, and HyperG. Cell morphology, proliferation, sphere formation, and differentiation potential were analyzed. Stem cell and tight junction markers were evaluated using flow cytometry, real-time PCR, Western blot, and immunofluorescence analysis. RESULTS: huMSGSCs showed fibroblast-like morphology and robust proliferation up to passage 10. Differentiation into adipocytes, chondrocytes, and osteocytes was successful, despite enhanced lineage-specific marker expression. HyperG significantly increased proliferation at 48 and 72 h, MicroG-exposed cells formed more numerous and smaller spheres, and HyperG-exposed cells produced larger spheres. HyperG elevated stem cell marker (CD90, LGR5, SOX2) expression levels, and the expression of tight junction protein expressions (ZO-1, ZO-2) was higher under HyperG treatment. CONCLUSIONS: Short-term HyperG and MicroG exposure differentially influenced huMSGSC stemness and differentiation potential. HyperG enhanced proliferation, stem cell marker expression, and differentiation capacity. These findings suggest the potential of optimizing huMSGSCs for regenerative therapies that target salivary gland dysfunction and other tissue regeneration applications.