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 assays showed that hemichannels opened as OGD progressed, peaking after 1 h, and then closed, returning to the pre-OGD state after 2 h of OGD. The increase in dye uptake after 1 h of OGD was inhibited by hemichannel blockers (lanthanum chloride and a connexin 43 mimetic peptide, Gap26). During OGD, intracellular Ca²⁺ concentration ([Ca²⁺]_i) began to increase after 1 h and reached several micromolar after 2 h. After 1 h of OGD, Gap26 inhibited the increases in hemichannel activity and [Ca²⁺]_i. In contrast, dantrolene [an endo(sarco)plasmic reticulum Ca²⁺ release inhibitor] suppressed the increase in [Ca²⁺]_i, but not in hemichannel activity. After 2 h 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 1 h of OGD. Simulated ischemia-reperfusion, induced by 1 h of OGD followed by 2 h 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) closure of hemichannels, but not their opening, is regulated by an increase in [Ca²⁺]_i during OGD, and 3) open hemichannels contribute to cell injury during recovery from OGD.