Osteoporosis is increasingly prevalent worldwide, representing a major health burden. However, there is a lack of nutritional strategies for osteoporotic therapy. Phytosterols, as natural bioactive compounds, have the potential to alleviate osteoporosis. In this study, a glucocorticoid-induced osteoporosis mouse model and treatment with low and high concentrations of phytosterols for 4 weeks were established. The results demonstrated that compared to the control group, low-concentration phytosterols (LP) (0.3 mg/mL) increased bone mass, improved trabecular microstructure, reduced serum levels of cross-linked C-telopeptide of type I collagen (CTX-1), and elevated serum levels of 1,25-dihydroxyvitamin D (1,25-(OH)D). Conversely, high-concentration phytosterols (0.5 mg/mL) showed no effect. Additionally, we validated the effect of LP in ameliorating osteoporosis using an ovariectomized (OVX)-induced osteoporosis mouse model. Mechanistically, phytosterols altered the microbial composition to counteract glucocorticoid-induced gut microbiota disorder and improve the length and morphology of the small intestine. Particularly, based on selection strategy and correlation analysis, phytosterols increased the relative abundance of and decreased the relative abundance of , which were significantly associated with glucocorticoid-induced osteoporosis indications. Overall, these findings suggest that phytosterols regulate gut microbiota to increase bone mass, thereby exerting an antiosteoporotic effect.