Anaerobic denitrification has been proved to be negatively affected by ZnO nanomaterials (NPs), but little is known about how ZnO NPs affects aerobic denitrification. In this study, inhibition of ZnO NPs to an aerobic denitrifier, Pseudomonas stutzeri PCN-1, was firstly reported. The results showed total nitrogen removal efficiency was decreased from 100% to 1.70% with the increase of ZnO NPs from 1 to 128mg/L. The presence of ZnO NPs caused significant inhibition of gene expressions and catalytic activities of nitrate reductase and nitrite reductase, which finally led to delayed nitrate reduction and high nitrite accumulation. Further studies revealed that the deposition of nanoparticles on the bacterial surface caused by electrostatic forces and the generation of reactive oxygen species (ROS) were responsible for the cytotoxicity of ZnO NPs, where ROS played a more important role. These results were of significance to evaluating the potential ecological toxicity and risks of nanomaterials.