A previous study showed that small G protein signaling modulator 3 (SGSM3) was highly correlated with Cx43 in heart functions and that high levels of SGSM3 may induce Cx43 turnover through lysosomal degradation in infarcted rat hearts. Here, we investigated the protective effects of kenpaullone on cardiomyocytes following H₂O₂-induced oxidative stress mediated by the interaction of SGSM3 with Cx43. We found that the gap junction protein Cx43 was significantly down-regulated in an H₂O₂ concentration-dependent manner, whereas expression of SGSM3 was up-regulated upon H₂O₂ exposure in H9c2 cells. The effect of kenpaullone pretreatment on H₂O₂-induced cytotoxicity was evaluated in H9c2 cells. H₂O₂ markedly increased the release of lactate dehydrogenase (LDH), while kenpaullone pretreatment suppressed LDH release in H9c2 cells. Moreover, kenpaullone pretreatment significantly reduced ROS fluorescence intensity and significantly down-regulated the level of apoptosis-activating genes (cleaved caspase-3, cleaved caspase-9 and cytochrome C), autophagy markers (LC3A/B), and the Cx43-interacting partner SGSM3. These results suggest that kenpaullone plays a role in protecting cardiomyocytes from oxidative stress and that the turnover of Cx43 through SGSM3-induced lysosomal degradation underlies the anti-apoptotic effect of kenpaullone.