The aim of this study was to investigate changes in hemichannel activity during in vitro simulated ischemia (oxygen-glucose deprivation, OGD) and the contribution of hemichannels to ischemia-reperfusion injury in rat neonatal cardiomyocytes. Dye uptake assay showed that hemichannels opened as OGD progressed, peaking after one hour, and then closed, returning to the pre-OGD state after 2 hours of OGD. The increase in dye uptake after one hour of OGD was inhibited by hemichannel blockers [lanthanum chloride (La³⁺) and a connexin 43 mimetic peptide, Gap26]. During OGD, intracellular Ca²⁺ concentration ([Ca²⁺]_i) began to increase after one hour and had reached a level of several µM after 2 hours. After one hour of OGD, Gap26 inhibited the increases in both hemichannel activity and [Ca²⁺]_i. In contrast, dantrolene (an ER/SR Ca²⁺-release inhibitor) suppressed the increase in [Ca²⁺]_i but not that in hemichannel activity. After 2 hours of OGD, the combined administration of 2’,4’-dichlorobenzamil and dantrolene reduced [Ca²⁺]_i to < 1 µM and increased hemichannel activity to the level attained after one hour of OGD. Simulated ischemia-reperfusion, afforded by one hour of OGD followed by 2 hours of recovery, reduced cell viability to 54% of the control level. The addition of Gap26 to OGD medium improved viability to 80% of the control level. In conclusion, this study demonstrated that 1) hemichannels open transiently during OGD, 2) the closure of hemichannels, but not their opening, is regulated by an increase in [Ca²⁺]_i during OGD, 3) open hemichannels contribute to cell injury during recovery from OGD.