In this study, nitrous oxide (N2O) production during biological nutrient removal (BNR) from municipal wastewater was reported to be remarkably reduced by controlling copper ion (Cu2+) concentration. Firstly, it was observed that the addition of Cu2+ (10-100 mu g/L) reduced N2O generation by 54.5-73.2 % and improved total nitrogen removal when synthetic wastewater was treated in an anaerobic-aerobic (with low dissolved oxygen) BNR process. Then, the roles of Cu2+ were investigated. The activities of nitrite and nitrous oxide reductases were increased by Cu2+ addition, which accelerated the bio-reductions of both nitrite to nitric oxide (NO (2) (-) -> aEuro parts per thousand NO) and nitrous oxide to nitrogen gas (N2O -> aEuro parts per thousand N-2). The quantitative real-time polymerase chain reaction assay indicated that Cu2+ addition increased the number of N2O reducing denitrifiers. Further investigation showed that more polyhydoxyalkanoates were utilized in the Cu2+-added system for denitrification. Finally, the feasibility of reducing N2O generation by controlling Cu2+ was examined in two other BNR processes treating real municipal wastewater. As the Cu2+ in municipal wastewater is usually below 10 mu g/L, according to this study, the supplement of influent Cu2+ to a concentration of 10-100 mu g/L is beneficial to reduce N2O emission and improve nitrogen removal when sludge concentration in the BNR system is around 3,200 mg/L.