Osteoporosis (OP) is one of the most common chronic metabolic bone diseases in the seniors and postmenopausal women. Plenty of microRNAs (miRNAs) have been confirmed to be involved in OP progression. However, the role of miR-655-3p in osteogenic differentiation and bone formation was still unclear. In this study, we aimed to investigate the cellular function of miR-655-3p and its underlying mechanism in OP. We found that miR-655-3p expression was downregulated in both ovariectomized (OVX) mice bone tissues and MC3T3-E1 cells treated with simulated microgravity (MG). MiR-655-3p overexpression facilitated cell differentiation but suppressed cell apoptosis of MC3T3-E1 cells induced by simulated MG. Mechanistically, we confirmed that lysine-specific histone demethylase 1 (LSD1) is a downstream target gene of miR-655-3p. Furthermore, overexpression of miR-655-3p activated the bone morphogenetic protein 2 (BMP-2)/decapentaplegic homolog (Smad) signaling pathway by suppressing LSD1 expression. Moreover, LSD1 knockdown accelerated osteogenic differentiation and inhibited apoptosis in MC3T3-E1 cells under simulated MG. Additionally, the OVX mouse model was established to investigate the role of miR-655-3p/LSD1 axis in vivo. The results demonstrated that LSD1 could reverse the effects triggered by the injection of adeno-associated virus-miR-655-3p on OP development. Further investigations revealed that miR-655-3p boosted osteogenic differentiation through LSD1/BMP-2/Smad signaling pathway. In summary, these findings implied a potential value of miR-655-3p in OP therapy.