Aim To investigate the role of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) in cardiomyocyte differentiation. Methods Anti-NOX4 ribozyme clones of mouse embryonic stem (ES) cells were differentiated into embryoid bodies (EBs). To measure reactive oxygenspecies (ROS) generation, H2DCF-DA and quantitative nitro blue tetrazolium (NBT) test was used. The mRNA levels of NOX were assayed by RT-PCR while the protein level of ventricular myosin light chain 2 (MLC2v) was detected by western blotting. And the apo ptosis of ES cells was detected by DNA laddering. Moreover, NOX4 mRNA in the heart of mouse embryo sections was analyzed by in situ hybridization. Results The exposure of embryoid bodies to 1-100 nmol/L H₂O₂ led to an enhanced beating activity, whereas 1 μmol/L H₂O₂ depressed cardiomyocyte differentiation as compared with control conditions (p<0.001). In contrast, ROS scavengers, such as N-acetylcysteine and catalase, and NOX inhibitor diphenyleneiodonium chloride impaired H₂O₂-stimulated cardiogenesis. The results revealed NOX4 as source of ROS responsible for ES cell differentiation into cardiomyocytes. Down-regulation of NOX4 expression in ES cells by ribozyme severely reduced ROS generation (p<0.001) and MLC2v expression, resulting in the suppression of cardiomyocyte differentiation (p<0.001). Moreover, NOX4 overexpression increased the production of ROS (p<0.05) and induced ES cell apo ptosis. Conclusion These results suggested a key role of NOX4 in cardiomyocyte differentiation through the generation of ROS.