The protein SET domain-containing lysine methyltransferase 7 (SETD7) has recently been shown to regulate apoptosis in various cells. However, the role of SETD7 on cardiomyocyte apoptosis during myocardial ischemia/reperfusion injury remains unclear. This study aimed to investigate the potential role of SETD7 in hypoxia/reoxygenation (H/R)-induced apoptosis of rat cardiomyocytes and reveal the underlying mechanism. Our results demonstrated that SETD7 expression was significantly up-regulated in cardiomyocytes in response to H/R injury. The inhibition of SETD7 by siRNA-mediated gene silencing significantly suppressed H/R-induced apoptosis and decreased the production of reactive oxygen species (ROS). The overexpression of SETD7 markedly enhanced H/R-induced apoptosis and ROS production. Moreover, the knockdown of SETD7 reduced the expression of Keap1 and promoted the expression of Nrf2. In addition, the knockdown of SETD7 increased the activity of antioxidant response element and promoted the expression of heme oxygenase-1 and NADPH-quinone oxidoreductase 1. However, the knockdown of Nrf2 partially abrogated the SETD7 inhibition-mediated protective effect against H/R injury. Taken together, these results indicate that the inhibition of SETD7 attenuates H/R-induced injury of cardiomyocytes via the down-regulation of Keap1 and promotion of the Nrf2-mediated anti-oxidation signaling pathway.