The release of nanoparticles (NPs) into the environment, including wastewater treatment plants, is expected to increase in the future. Therefore, it is important to understand the potential effects of these NPs on activated sludge treatment processes. A pulse-flow respirometer was used to study the toxicity of nano-ZnO on activated sludge endogenous respiration, BOD biodegradation, and nitrification. In addition, toxicities of bulk ZnO particles and Zn ion (e.g. soluble Zn) were also studied. All three Zn forms were found to adversely impact the activity of activated sludge, with soluble Zn exhibited the greatest toxicity. The effects of nano-ZnO and bulk ZnO on activated sludge were caused by soluble Zn resulting from ZnO particle dissolution. The IC50 values of soluble Zn on activated sludge endogenous respiration, BOD biodegradation, ammonia oxidation, and nitrite oxidation were 2.2, 1.3, 0.8, and 7.3 mg-Zn/L, respectively. Therefore, the first step of nitrification was most sensitive to Zn.