A superoxide-generating NADPH oxidase (NOX) has been reported to mediate cytotoxicity induced by cigarette smoke components. However, the underlying molecular mechanisms and NOX isoform involved have not been fully clarified. Among NOX isoforms identified so far, NOX1 and NOX4 were expressed in rat H9c2 cardiomyocytes. When H9c2 cells were exposed to acrolein or methyl vinyl ketone (MVK), major toxic components of cigarette smoke extracts, a dose-dependent decline in cell viability was observed. Unexpectedly, disruption of Nox1 significantly exacerbated cytotoxicity induced by acrolein or MVK. The levels of total and reduced glutathione (GSH) were significantly reduced in Nox1-disrupted H9c2 clones. In these clones, expression of multidrug resistance-associated protein 1 (MRP1), which mediates glutathione efflux, was significantly up-regulated. Treatment of reversan, an MRP1 inhibitor, partially but significantly blunted the cytotoxicity of acrolein and MVK in Nox1-disrupted cells. Taken together, NOX1/NADPH oxidase regulates the expression of MRP1 to maintain intracellular GSH levels in cardiomyocytes and protect against cytotoxic components of cigarette smoke extracts.