In this study, four reactors named as R1 (control reactor), R2 (fed with 40 nm CuO-NPs), R3 (fed with 200 nm CuO-NPs), and R4 (fed with Cu(II)) were adopted to investigate the influence of CuO-NPs with different particle size on the nitrogen removal and microbial community of the Anaerobic ammonium oxidation (Anammox) process. Compared with the control reactor, the total nitrogen removal rate decreased to 0.2 and 0.43 kg/(m3·d) in R2 and R3, respectively, but it increased from 0.63 kg/(m3·d) to 0.77 kg/(m·d) in R4. Proportions of anaerobic ammonia-oxidizing bacteria R4 (12.88%) >R1 (12.37%) >R3 (8.03%) >R2 (2.52%). Results indicated that Cu(II) promoted Anammox bioactivity, whereas CuO-NPs had a significant inhibitory effect; the NPs with smaller size exhibited more severe effects, whereas the results verified that the suppression of Anammox by CuO-NPs was not due to the released Cu(II), but the nanoscale effect of the NPs, and the smaller particle size exhibited a more severe impact. [ABSTRACT FROM AUTHOR]