Although nitric oxide (NO) emissions from anaerobic ammonium oxidation (anammox)-based processes were reported previously, the NO production pathways are poorly understood. Here, we investigated the NO production pathways in anammox granules in detail by combining 15N-stable isotope tracer experiments with various inhibitors, microsensor measurements, and transcriptome analysis for key genes of NO2- reduction. NO was emitted from the anammox granules, which account for 0.07% of the N2 emission. 15N-stable isotope-tracer experiments indicated that most of the N2 was produced by anammox bacteria, whereas NO was produced from NO2- reduction by anammox and denitrifying bacteria. The NO emission rate was highest at pH 8.0 and accelerated by increasing NH4+ and NO2- concentrations in the culture media. The microsensor analyses showed the in situ NO production rate was highest in the outer layer of the anammox granule where anammox activity was also highest. The detected in situ NO concentrations of up to 2.7 μM were significantly above physiological thresholds known to affect a wide range of microorganisms present in wastewater. Hence, NO likely plays pivotal roles in the microbial interactions in anammox granules, which needs to be further investigated.