BACKGROUND The incidence of osteoporosis is positively correlated with age. Berberine has been reported to treat osteoporosis due to its beneficial actions on bone formation. However, the direct effects of berberine on senile osteoporosis remain unclear. The present study investigated the protective effects of berberine on senile osteoporosis in mice and preliminarily evaluated its potential mechanism. METHODS 20-month-old male C57BL/6 J mice were used as senile osteoporosis mouse model and treated with strontium ranelate (SR) or berberine or solvent control by daily gavage for 2 months. Thereafter, bone mass and microstructure parameters were assessed. Histological staining was performed to identify the osteogenic, adipogenic and osteoclastic activity of bone tissue. Moreover, role of cAMP/PKA/CREB signaling pathway in berberine affecting bone marrow mesenchymal stem cells (BMSCs) differentiation was clarified by enzyme-linked immunosorbent assay and western blot analysis. RESULTS The results showed that the SR-treated group displayed a high trabecular bone mass phenotype. For mice administrated with berberine, cancellous bone mass was upregulated in a dose-dependent manner, as indicated by gradually increased bone mass, trabecular bone volume fraction and trabecular number. Furthermore, berberine promotes osteogenic and inhibits adipogenic differentiation of BMSCs via cAMP/PKA/CREB signaling. Also, bone resorption effect becomes more obvious with increasing dose of berberine in vitro. CONCLUSIONS The present results suggest that berberine exerts potent bone protective effects by promoting bone formation, inhibiting marrow fat accumulation and bone resorption. This effect may be achieved through cAMP/PKA/CREB signaling pathway.