Nitrification in wastewater treatment emits a significant amount of nitrous oxide (N2O), which is one of the major greenhouse gases. However, the actual mechanism or metabolic pathway is still largely unknown. Selective nitrification inhibitors were used to determine the nitrification steps responsible for N2O emission with activated sludge and enriched nitrifiers. Allylthiourea (86 μM) completely inhibited ammonia oxidation and N2O emission both in activated sludge and enriched nitrifiers. Sodium azide (24 μM) selectively inhibited nitrite oxidation and it led to more N2O emission than the control experiment both in activated sludge and enriched nitrifiers. The inhibition tests showed that N2O emission was mainly related to the activity of ammonia oxidizers in aerobic condition, and the inhibition of ammonia monooxygenase completely blocked N2O emission. On the other hand, N2O emission increased significantly as the nitrogen flux from nitrite to nitrate was blocked by the selective inhibition of nitrite oxidation.