Background <p>Glucocorticoid-induced osteoporosis (GIOP) represents the predominant type of secondary bone loss disorder, linked to a substantially elevated incidence of pathological fractures in clinical populations. Modified Yanghe Decoction (MYHD), a classical Chinese medicinal formula, has demonstrated therapeutic potential in managing osteoporotic conditions. Nevertheless, the specific mechanisms underlying MYHD’s effectiveness against GIOP require further elucidation, posing challenges for broader clinical implementation.</p> Methods <p>We performed comprehensive analysis of bioactive constituents in the MYHD n-butanol (MYHD-NB) fraction through high-performance liquid chromatography, followed by mechanistic exploration of its biological effects across experimental models. Ten-week-old male C57BL/6J mice received dexamethasone (DEX)-induced modeling and were administered either MYHD or varying concentrations of MYHD-NB via oral gavage. Following an 8-week intervention, femoral specimens underwent multimodal evaluation including histopathological examination, micro-computed tomography imaging, and advanced molecular detection techniques (immunohistochemistry/immunofluorescence). Parallel bioinformatics analysis was executed on RNA-seq datasets from osteoporotic fracture patients in the GEO repository. For in vitro experimentation, DEX-treated murine BMSCs were employed to assess MYHD-NB’s regulatory effects on cellular proliferation, migratory capacity, osteogenic differentiation potential, and ferroptotic processes. Complementary network pharmacology approaches were utilized to predict potential molecular targets and pathway interactions.</p> Results <p>MYHD-NB significantly improved the osteoporosis-like phenotype in mice, as indicated by the improvement of bone microstructure and the upregulation of ALP and RUNX2 expression. RNA-seq analysis of the GEO dataset showed that ferroptosis-related genes are significantly upregulated in the femurs of GIOP patients. Both in vivo and in vitro studies confirmed MYHD-NB’s capacity to mitigate dexamethasone-induced ferroptosis in BMSCs while enhancing BMSCs proliferation capacity, migratory potential, and differentiation into osteogenic lineages. Network pharmacology predictions identified HIF-1α as the pivotal regulator mediating MYHD-NB’s anti-ferroptotic effects. Molecular investigations revealed MYHD-NB’s activation of the HIF-1α/GPX4 signaling pathway, with genetic ablation of HIF-1α completely negating the compound’s therapeutic efficacy.</p> Conclusion <p>MYHD-NB mitigates ferroptosis triggered by DEX in BMSCs while enhancing osteogenic differentiation via the HIF-1α/GPX4 signaling pathway, elucidating its therapeutic mechanism against GIOP. These results demonstrate MYHD-NB’s superior osteogenic potential within the MYHD formulation, positioning it as a promising candidate for alternative GIOP management strategies.</p>