This study provides a comprehensive evaluation of the anti-osteoporotic effects of flavonoids derived from Epimedium, including icaritin and its six glycosides-icariside I, icariside II, icariin, epimedin A, epimedin B, and epimedin C-using a zebrafish model of iron overload-induced osteoporosis. Our results demonstrate a significant increase in lipid peroxidation in zebrafish subjected to ferric ammonium citrate (FAC)-induced osteoporosis, along with impaired expression and activity of glutathione peroxidase 4 (GPX4). Treatment with ferrostatin-1, a lipid peroxide scavenger, partially alleviated the osteoporotic effects induced by FAC, implying that lipid peroxidation may play a key role in iron overload-related osteoporosis. We observed varying degrees of anti-osteoporotic activity and enhancement of osteogenic differentiation markers, such as bmp2b, runx2b, col1a1a, and alp, among icaritin and its glycosides. Notably, icaritin exhibited the most potent inhibitory effects on osteoporosis, while epimedin A and epimedin B showed enhanced efficacy compared to other glycosides, correlating closely with their ability to suppress lipid peroxidation. Additionally, through CETSA, molecular docking, and dynamic simulation studies, we identified an interaction between icaritin and GPX4, which may help stabilizing GPX4 against FAC-induced lipid peroxidation. These findings suggest that the anti-osteoporotic effects of icaritin and its glycosides are linked to their ability to suppress lipid peroxidation, offering potential therapeutic insights for managing iron overload-induced osteoporosis.