--- - |2- Abstract Dual‐specificity protein phosphatases (DUSP) also known as mitogen‐activated protein kinase (MAPK) phosphatases (MKPs) can dephosphorylate MAPKs, including extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase (JNK), and p38. DUSP1‐mediated JNK dephosphorylation has been found to play an antiapoptotic role against cardiac ischemia–reperfusion (I/R) injury. However, the regulation of DUSP1–JNK pathway remains unclear. In the current study, ubiquitin‐specific peptidase 49 (USP49) expression in human AC16 cardiomyocytes following I/R injury was measured by real‐time polymerase chain reaction and western blot analysis. Cell viability, apoptosis, the Bax, Bcl‐2, and DUSP1 expression, and the activity of MAPKs in AC16 cardiomyocytes following indicated treatment was measured by CCK‐8, flow cytometry, and western blot analysis. The direct interaction between USP49 and DUSP1 was measured by coimmunoprecipitation and ubiquitination analysis. The effect of USP49 on apoptosis and JNK activity in rat cardiomyocytes following I/R injury was also measured by TUNEL and western blot analysis. Here, we found that USP49 expression was time‐dependently increased in AC16 cardiomyocytes following I/R. I/R‐induced cell apoptosis and JNK1/2 activation both in in vivo and in vitro reversed by USP49 overexpression in AC16 cardiomyocytes. Inhibiting JNK1/2 activation significantly inhibited USP49 knockdown‐induced the cell viability inhibition, apoptosis and the JNK1/2 activation in AC16 cardiomyocytes. Moreover, USP49 positively regulated DUSP1 expression through deubiquitinating DUSP1. Overall, our findings establish USP49 as a novel regulator of DUSP1–JNK1/2 signaling pathway with a protective role in cardiac I/R injury. - Journal of Cellular Physiology, EarlyView.