Aim To elucidate the change of intracellular reactive oxygen species (ROS) level and apoptosis in human umbilical vein endothelial cells (hUVEC and ECV304) after transfection with pcDNA3.1-nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) plasmid. Methods The level of NOX4 mRNA in hUVEC and ECV304 transfected with pcDNA3.1-NOX4 plasmid or pcDNA3.1-GFP plasmid was measured by RT-PCR. Intracellular ROS level and apoptosis rate were detected with flow cytometry. The apoptosis of hUVEC and ECV304 was observed by Hoechst staining and TUNEL staining. Results NOX4 mRNA expression and ROS level were increased significantly in hUVEC transfected with pcDNA3.1-NOX4 plasmid compared with control group and the group that were transfected with pcDNA3.1-GFP plasmid (p<0.05,n=3). NOX4 mRNA was also detectable in ECV304 transfected with pcDNA3.1-NOX4 plasmid, but not in control ECV304. The apoptosis rate were elevated significantly in hUVEC and ECV304 transfected with pcDNA3.1-NOX4 plasmid (the apoptosis rate of ECV304 and hUVEC is 9.6%±0.9% and 12.4%±1.1% respectively) compared with other two groups (the apoptosis rate of ECV304 and hUVEC in control group is 1.05±0.25%, 2.28±0.37% respectively, the apoptosis rate of ECV304 and hUVEC in the group that were transfected with pcDNA3.1-GFP plasmid is 1.56%±0.33% and 4.56%±0.62% respectively) as detected by flow cytometry (p<0.05, n=3). The results of Hoechst staining and TUNEL staining indicated that apoptotic cells present in hUVEC and ECV304 transfected with pcDNA3.1-NOX4 plasmid,but not in other two groups. Conclusion The transfection of plasmid pcDNA3.1-NOX4 results in over-expression of NOX4 in hUVEC and ECV304,inducing apoptosis of hUVEC and ECV304.