An increase of nitrite in the domestic-strength range is generally recognized to stimulate nitrous oxide (N2O) production by ammonia-oxidizing bacteria (AOB). It was found in this study, however, that N2O emission from a mainstream nitritation system (cyclic nitrite = 25-45 mg of N/L) that was established by free nitrous acid (FNA)-based sludge treatment was not higher but much lower than that from the initial nitrifying system with full conversion of NH4(+)-N to NO3(-)-N. Under dissolved oxygen (DO) levels of 2.5-3.0 mg/L, N2O emission from the nitritation stage was 76% lower than that from the initial stage. Even when the DO level was reduced to 0.3-0.8 mg/L, N2O emission from the nitritation stage was still 40% lower. An investigation of the mechanism showed that FNA treatment caused a shift of the stimulation threshold of nitrite on N2O emission. At the nitritation stage, the maximal N2O emission factor occurred at ∼16 mg of N/(L of nitrite). However, it increased with increasing nitrite in the range of 0-56 mg of N/L at the initial stage. FNA treatment decreased the biomass-specific N2O production rate, suggesting that the enzymes relevant to nitrifier denitrification were inhibited. Microbial analysis revealed that FNA treatment decreased the microbial community diversity but increased the abundances of AOB and denitrifiers.