The sustainability of autotrophic granular system performing partial nitritation and anaerobic ammonium oxidation (anammox) for complete nitrogen removal is impaired by the production of nitrous oxide (N2O). A systematic analysis of the pathways and affecting parameters is, therefore, required for developing N O-2 mitigation strategies. To this end, a mathematical model capable of describing different N O-2 production pathways was defined in this study by synthesizing relevant mechanisms of ammonium-oxidizing bacteria (AOB), nitrite-oxidizing bacteria, heterotrophic bacteria (HB), and anammox bacteria. With the model validity reliably tested and verified using two independent sets of experimental data from two different autotrophic nitrogen removal biofilm/granular systems, the defined model was applied to reveal the underlying mechanisms of N O-2 production in the granular structure as well as the impacts of operating conditions on N O-2 production. The results show that: (a) in the aerobic zone close to the granule surface where AOB contribute to N O-2 production through both the AOB denitrification pathway and the NH 2OH pathway, the co-occurring HB consume N O-2 produced by AOB but indirectly enhance the N O-2 production by providing NO from NO (2) (-) reduction for the NH 2OH pathway, (b) the inner anoxic zone of granules with the dominance of anammox bacteria acts as a sink for NO (2) (-) diffusing from the outer aerobic zone and, therefore, reduces N O-2 production from the AOB denitrification pathway, (c) operating parameters including bulk DO, influent NH (4) (+), and granule size affect the N O-2 production in the granules mainly by regulating the NH 2OH pathway of AOB, accounting for 34-58% of N O-2 turnover, and (d) the competition between the NH 2OH pathway and heterotrophic denitrification for nitric oxide leads to the positive role of HB in reducing N O-2 production in the autotrophic nitrogen removal granules, which could be further enhanced in the presence of a proper level of influent organics.