Connexin 43 (Cx43), known to be the main protein building blocks of gap junctions and hemichannels in mammalian heart, plays an important role in cardiocytes proliferation. Gap junctional intercellular communication has been suggested to be necessary for cellular proliferation and differentiation. However, the effect of Cx43 hemichannel on cardiocytes proliferation and the mechanism remain unclear. In this study, rat heart cell line H9c2 was used. The Cx43 location, the proliferation rate and hemichannel activity of H9c2 cells and Wnt-3a(+)-H9c2 cells were investigated and the changes of intracellular ATP and [Ca(2+)] were determined. Results showed that the inhibited hemichannel induced by 18beta-glycyrrhetinic acid (GA) evoked intracellular ATP and [Ca(2+)] increase and enhanced H9c2 cell proliferation. Wnt-3a(+)-H9c2 cells displayed enhanced hemichannel activity and proliferation rate. Inhibited hemichannel of Wnt-3a(+)-H9c2 cells induced by 18beta-GA decreased intracellular ATP, increased [Ca(2+)], and enhanced the proliferation of H9c2 cells. This study validated the role of hemichannel in H9c2 cell proliferation regulation, and showed a mechanism involved in the regulation of H9c2 cell proliferation. The proliferation could be enhanced by Cx43 hemichannel-mediated ATP release accompanying intracellular [Ca(2+)] change. However, different changes of ATP were observed in Wnt-3a(+)-H9c2 cells. These findings provided new insights into the molecular mechanisms of proliferation regulation in H9c2 cells and the effect of Wnt-3a on intracellular ATP.