Abstract: AIM:To investigate the role of NAD(P)H oxidase 4 (Nox4) in cardiomyocytes exposed to hypoxia/reoxygenation and its potential mechanism. METHODS: H9C2 myocytes were exposed to hypoxia/reoxygenation (H/R) and Nox4 was downregulated by RNA interference. H9C2 myocytes were divided into control group, NC-siRNA group, Nox4-siRNA group, H/R group, H/R+NC-siRNA group and H/R+Nox4-siRNA group. Cell viability was detected by MTT assay. Cellular ATP level was measured by luciferase chemiluminescence assay and NAD+/NADH was assessed by a commercial assay kit followed by colorimetry. Mitochondrial ROS production was detected by MitoSOX fluorescent probe and protein expression of Nox4 and SIRT3 was analyzed by Western blot. RESULTS: Compared with control group, H/R induced increased protein expression of Nox4 and resulted in significant reduction in cell viability and ATP level with a remarkable increase in mitochondrial ROS production. NAD+/NADH ratio increased, whereas SIRT3 protein expression decreased in response to H/R. Compared with H/R group, H/R+Nox4-siRNA group exhibited further decline in cell viability and ATP level, with enhanced mitochondrial ROS production. More importantly, the increased NAD+/NADH following H/R was reversed by Nox4 inhibition, whereas SIRT3 protein expression was further suppressed. CONCLUSION: Downregulation of Nox4 exacerbates H/R-induced cardiomyocyte injury by increasing mitochondrial ROS production and inhibiting mitochondrial energetics. The protective role of Nox4 against H/R injury may be attributed to the upregulated NAD+/NADH-SIRT3 signaling.