Objective To investigate whether extracorporeal shockwave could induce differentiation of human mesenchymal stem cells (hMSCs) into osteoprogenitor cells by ATP release and the activation of P2X7 receptors. Methods Cultured bone marrow-derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase (ALP) activity, osteocalcin (OC) production, and calcium nodule formation. The mRNA expression of P2X7 receptors was determined with real-time RT-PCR. P2X7-siRNA, apyrase, and P2 receptor antagonists were used to evaluate the roles of ATP release and P2X7 receptors in shockwave-induced osteogenic hMSCs differentiation. Results Shockwave treatment released significant amounts of ATP from hMSCs. Shockwaves and exogenous ATP induced hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2X7-siRNA or selective antagonists prevented osteogenic differentiation of hMSCs. Conclusions Shockwaves can contribute to osteogenic differentiation of hMSCs by realeasing cellular ATP that activate signaling. These research findings provide the theoretical basis for shockwave therapy in treating fracture healing and bone nonunion.