Microgravity (μXg) experienced by astronauts during space flights causes accelerated bone loss. However, the molecular basis of μXg induced bone loss in space is unclear. Osteoclast (OCL) is the primary bone-resorbing cell. We previously demonstrated that simulated μXg promotes OCL formation. In this study, we identified that μXg induces syncytin-A expression in RAW264.7 preosteoclast cells without RANKL stimulation. We further tested the effect of osteotropic factors such as CXCL5 and 1,25(OH) D to regulate the syncytin-A expression in preosteoclast cells subjected to μXg compared to ground based (Xg) cultures. CXCL5 (25 ng/mL) and 1,25(OH) D (10 ng/mL) increased syncytin-A expression under Xg conditions. However, μXg alone upregulates syncytin-A expression compared to Xg control preosteoclast cells. Confocal microscopy using Lyso-Tracker identified syncytin-A expression co-localized with lysosomes in preosteoclast cells. Acridine orange staining showed RANKL elevated autophagy activity in these cells. Further, siRNA suppression of syncytin-A significantly inhibits autophagy activity in RAW264.7 cells. In addition, knockdown of syncytin-A expression inhibits μXg increased OCL formation in mouse bone marrow cultures. Thus, our findings suggest that targeting syncytin-A expression may be an effective countermeasure to control bone loss under microgravity conditions.