Objective To investigate the role of miR-194-3p in regulating functional changes in osteoblasts in a simulated microgravity environment and to provide a theoretical foundation for understanding the mechanical response mechanisms of osteoblasts in extreme mechanical environments. Methods The effects of microgravity on osteoblasts were simulated by using a rotary cell culture system. MC3T3-E1 osteoblasts were transfected with an miR-194-3p inhibitor and changes in proliferation, differentiation, apoptosis, and mineralization were assessed using MTT assay, RT-PCR, western blotting, double fluorescence staining, and alizarin red staining. Results Elevated expression of miR-194-3p under simulated microgravity conditions led to the suppression of osteoblast proliferation, differentiation, and mineralization to a certain extent, while promoting osteoblast apoptosis. However, transfection with the miR-194-3p inhibitor significantly downregulated miR-194-3p expression and partially reversed the reduced osteoblast proliferation, decreased expression of osteogenic differentiation markers such as ALP, OCN, and COL-I genes and proteins, decreased bone mineralization nodules, and increased osteoblast apoptosis induced by microgravity exposure. These findings indicated that miR-194-3p effectively ameliorates abnormal osteoblast function under microgravity conditions. Conclusions MiR-194-3p acted as a negative regulatory factor in the mechanical responses of osteoblasts under simulated microgravity.