Oxidative stress is a major cause of I/R injury. NADPH oxidase is an important source of oxidative stress during I/R, and it consists of NOX1-5 and DOUX1-2 isoforms. The role of NOX1 in myocardial I/R injury is unclear. In this study, a specific NOX1 inhibitor, ML171 (MW=241.31 g/mol, IC50 for NOX1=0.25 μM), was tested in isolated perfused rat hearts following I (30 min)/R (45 min). We found that left ventricular developed pressure (LVDP) and the peak of the first derivative of left ventricular pressure (dP/dtmax) only recovered to 42 ± 4% and 30 ± 3% of baseline values, respectively, at 45 min post-reperfusion in control I/R hearts (n=8). By contrast, ML171 (1 μM, n=5) given at reperfusion for 5 min significantly restored LVDP and dP/dtmax to 90 ± 6% and 67 ± 7% of baseline values, respectively (both p<0.01). ML171 also significantly reduced infarct size to 18 ± 3% compared to 41 ± 2% in control I/R hearts (p<0.01). Furthermore, a selective mitochondrial ATP-dependent K+ channel (mitoKatp) inhibitor, 5- hydroxydecanoate (100 μM, n=4); and a heme oxygenase-1 inhibitor, SnPP (20 μM, n=3), significantly abolished the cardioprotective effects of ML171 (all p<0.05). We also confirmed that ML171 (0.1-1 μM, n=4) did not inhibit phorbol 12-myristate 13-acetate (30 nM) induced superoxide release from isolated rat neutrophils. These results suggest that NOX1 inhibition improves post-reperfused contractile function and reduces infarct size, possibly by opening mitoKatp channels and activating heme oxygenase-1.