Aims. In our previous research, we have found that melatonin (MEL) affects the osteoporotic process. By balancing bone remoulding, autophagy is involved in age-related bone loss. However, as a regulator of autophagy, whether MEL influences senile osteoporosis via regulating autophagy remains unclear. Methods. Cellular, radiological, and histopathological evaluations were performed on 36 16-month-old male C57BL6/L mice or aged bone marrow-derived mesenchymal stem cells. A MEL-gelatin methacrylamide system was constructed to aid osteoporotic fracture healing. Results. In this study, we found that bone loss, low level of MEL, and decreased autophagy coexisted in aged C57BL6/L mice. A physiological (low, 10 nM but not 100 nM) concentration of MEL restored bone loss, transformed the cytokine framework, and increased the autophagic level in aged mice, whereas inhibition of autophagy unfavourably reduced the positive effects of MEL on bone mass. The autophagy-conducted increased osteogenic lineage commitment and extracellular matrix mineralization, but not matrix synthesis of aged bone marrow-derived mesenchymal stem cells, was responsible for MEL anabolic effects on bone. PIK3C-AKT-MTOR signal was tested to be a main pathway that is involved in MEL-induced autophagy. Conclusion. Our data suggest that the application of MEL can restore degenerative osteogenesis of aged bone marrow-derived mesenchymal stem cells, and has the potential to regain bone mass in aged mice through activating autophagy via the PIK3C-AKT-MTOR pathway. MEL therefore may serve as a potential clinical therapy to treat senile osteoporosis. Cite this article: Bone Joint Res 2025;14(2):97–110