Luteolin (LUT) has been suggested as an inhibitor of osteoporosis (OP). This investigation examines the pivotal role of the miR-125b-5p/SIRT3/AMPK/mTOR pathway in mediating luteolin-induced effects on OP. Mesenchymal stem cells derived from bone marrow (BMSCs) were exposed to dexamethasone (DEX) to create an in vitro model of OP. Following treatment with luteolin, the levels of miR-125b-5p and SIRT3 were quantified using reverse transcription polymerase chain reaction. Moreover, SIRT3, AMPK, mTOR protein levels, and osteogenesis (OPN, Runx2, OSX, and OCN), and autophagy (p62, ATG5, LC3, and BECN1) were evaluated using ELISA. Additionally, specific mimics and siRNA were constructed to overexpress miR-125b-5p or downregulate SIRT3. Furthermore, animal models of DEX-induced OP were constructed to assess the effects of LUT at doses of 50 and 100 mg/kg/day on bone histology, stereology, biochemistry, and the expression of the miR-125b-5p, SIRT3/AMPK/mTOR axis, and markers of osteogenesis and autophagy. The findings revealed that LUT suppressed miR-125b-5p expression, overexpressed SIRT3 and AMPK, and downregulated mTOR in BMSCs compared to DEX (p-value < 0.01). Interestingly, LUT restored the levels of markers for osteogenesis and autophagy (p-value < 0.001). The overexpression of SIRT3 or miR-125b-5p downregulation inhibited LUT therapeutic properties. In animals, LUT improved bone histology (p-value < 0.05) and inhibited miR-125b-5p and mTOR expression while overexpressing SIRT3 and AMPK (p-value < 0.001). RUNX2, OSX, OPN, and OCN levels were improved, and autophagy was enhanced in LUT-treated rats. The current findings revealed that LUT could promote osteogenesis and improve OP via autophagy activation through the miR-125b-5p/SIRT3/AMPK/mTOR pathway.© 2025 The Author(s). Food Science & Nutrition published by Wiley Periodicals LLC.