The key pathological process of osteoporosis is the imbalance of bone remodeling. However, the detailed mechanism of bone homeostasis imbalance leading to osteoporosis is poorly understood. To explore the core proteins and potential regulatory pathways associated with severe osteoporosis, bone specimens were obtained during surgery from 13 women with osteoporotic fractures and 8 controls with violent fractures, along with 12 men with osteoporotic fractures and 7 controls with violent fractures. Non-targeted proteomics analysis based on liquid chromatography-tandem mass spectrometry showed differentially abundant proteins (DAPs) of women with osteoporotic fractures were mainly enriched in pathways such as phagosome, lysosome, integrin-mediated cell adhesion and antigen processing and presentation, primarily related to immunoinflammatory response, while DAPs of men with osteoporotic fractures were mainly enriched in pathways including oxidative phosphorylation, reactive oxygen species, mitochondrial respiratory chain complex I assembly, and mitochondrial electron transport and cellular respiration, primarily related to oxidative stress response. ROC analysis revealed relative strong associations between core upregulated proteins and osteoporosis in both women (AUC = 0.903, 95% CI: 0.718-1) and men (AUC = 0.851, 95% CI: 0.366-1). These proteins were negatively correlated with BMD (P<0.05) and positively correlated with β-CTX or P1NP (P<0.05). Validation of core differential proteins in additional bone specimens showed HLA-C expression was significantly higher in women with osteoporotic fractures than in the control group (P<0.05). ITGB2 exhibited a trend of higher expression in women with osteoporosis than in the control group, while COX7A2 showed a similar trend of higher expression in men with osteoporotic fractures than in the control group. This study identified distinct proteomic expression profiles and molecular pathways of osteoporosis in women and men, which helps to understand the gender differences in the pathogenesis of osteoporosis and provides a foundation for discovering new therapeutic targets and developing personalized precision treatment strategies.