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Bone marrow‐derived human mesenchymal stem cells (hMSCs) have the potential to differentiate into several cell lines. Extracellular adenosine 5′‐triphosphate (ATP) acts as a potent signaling molecule mediating cell‐to‐cell communication. Particular interest has been focused in recent years on the role of ATP in stem cell proliferation and differentiation. In the present work, we demonstrate that hMSCs at early stages of culture (P0–P5) spontaneously release ATP, which decreases cell proliferation. Increased hMSC proliferation is induced by the unselective P2 antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonate (PPADS) and by the selective P2Y₁ antagonist 2′‐deoxy‐N⁶‐methyladenosine3′,5′‐bisphosphate (MRS 2179). A functional role of extracellular ATP in modulating ionic conductances with the whole‐cell and/or perforated patch‐clamp techniques was also investigated …