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Background and Purpose: Caffeine is consumed extensively in Europe and North America. As a risk factor for osteoporosis, epidemiological studies have observed that caffeine can decrease bone mineral density, adversely affect calcium absorption and increase the risk of bone fracture. However, the exact mechanisms have not been fully investigated. Here, we examined the effects of caffeine on the viability and osteogenesis of rat bone marrow-derived mesenchymal stromal cells (rBMSCs)., Experimental Approach: Cell viability, apoptosis and necrosis were quantified using thymidine incorporation and flow cytometry. Sequential gene expressions in osteogenic process were measured by real-time PCR. cAMP, alkaline phosphatase and osteocalcin were assessed by immunoassay, spectrophotometry and radioimmunoassay, respectively. Mineralization was determined by calcium deposition., Key Results: After treating BMSCs with high caffeine concentrations (0.1-1mM), their viability decreased in a concentration-dependent manner. This cell death was primarily due to necrosis and, to a small extent, apoptosis. Genes and protein sequentially expressed in osteogenesis, including Cbfa1/Runx2, collagen I, alkaline phosphatase and its protein, were significantly downregulated except for osteocalcin and its protein. Moreover, caffeine inhibited calcium deposition in a concentration- and time-dependent manner, but increased intracellular cAMP in a concentration-dependent manner., Conclusions and Implications: By suppressing the commitment of BMSCs to the osteogenic lineage and selectively inhibiting gene expression, caffeine downregulated some important events in osteogenesis and ultimately affected bone mass.