The influences of cerium dioxide nanoparticles (CeO2 NPs) on nitrogen removal in biofilm were investigated. Prolonged exposure (75 d) to 0.1 mg/L CeO2 NPs caused no inhibitory effects on nitrogen removal, while continuous addition of 10 mg/L CeO2 NPs decreased the treatment efficiency to 53%. With the progressive concentration of CeO2 NPs addition, the removal efficiency could nearly stabilize at 67% even with the continues spike of 10 mg/L. The micro-profiles of dissolved oxygen, pH, and oxidation reduction potential suggested the developed protection mechanisms of microbes to progressive CeO2 NPs exposure led to the less influence of microenvironment, denitrification bacteria and enzyme activity than those with continuous ones. Furthermore, high throughput sequencing illustrated the drastic shifted communities with gradual CeO2 NPs spiking was responsible for the adaption and protective mechanisms. The present study demonstrated the acclimated microbial community was able to survive CeO2 NPs addition more readily than those non-acclimated.