Dissolved oxygen (DO) is commonly recognized as an important factor influencing nitrous oxide (N2O) production by ammonia-oxidizing bacteria (AOB). However, it has been difficult to separate the true effect of DO from that of nitrite, as DO variation often affects nitrite accumulation. The effect of DO on N2O production by an enriched nitrifying sludge, consisting of both AOB and nitrite-oxidizing bacteria (NOB), was investigated in this study. Nitrite accumulation was minimized by augmenting nitrite oxidation through the addition of an enriched NOB sludge. It was demonstrated that the specific N2O production rate increased from 0 to 1.9 ± 0.09 (n = 3) mg N2O–N/hr/g VSS with an increase of DO concentration from 0 to 3.0 mg O2/L, whereas N2O emission factor (the ratio between N2O nitrogen emitted and the ammonium nitrogen converted) decreased from 10.6 ± 1.7% (n = 3) at DO = 0.2 mg O2/L to 2.4 ± 0.1% (n = 3) at DO = 3.0 mg O2/L. The site preference measurements indicated that both the AOB denitrification and hydroxylamine (NH2OH) oxidation pathways contributed to N2O production, and DO had an important effect on the relative contributions of the two pathways. This finding is supported by analysis of the processed data using an N2O model describing both pathways. As DO increased from 0.2 to 3.0 mg O2/L, the contribution of AOB denitrification decreased from 92% – 95%–66% – 73%, accompanied by a corresponding increase in the contribution by the NH2OH oxidation pathway.