Nitrous oxide (N2O) is a key climate change gas and nitrifying microbes living in terrestrial ecosystems contribute significantly to its formation. Many soils are acidic and global change will cause acidification of aquatic and terrestrial ecosystems, but the effect of decreasing pH on N2O formation by nitrifiers is poorly understood. Here, we used isotope-ratio mass spectrometry to investigate the effect of acidification on production of N2O by pure cultures of two ammonia-oxidizing archaea (AOA; Nitrosocosmicus oleophilus and Nitrosotenuis chungbukensis) and an ammonia-oxidizing bacterium (AOB; Nitrosomonas europaea). For all three strains acidification led to increased emission of N2O. However, changes of 15N site preference (SP) values within the N2O molecule (as indicators of pathways for N2O formation), caused by decreasing pH, were highly different between the tested AOA and AOB. While acidification decreased the SP value in the AOB strain, SP values increased to a maximum value of 29‰ in N. oleophilus. In addition, 15N-nitrite tracer experiments showed that acidification boosted nitrite transformation into N2O in all strains, but the incorporation rate was different for each ammonia oxidizer. Unexpectedly, for N. oleophilus more than 50% of the N2O produced at pH 5.5 had both nitrogen atoms from nitrite and we demonstrated that under these conditions expression of a putative cytochrome P450 NO reductase is strongly upregulated. Collectively, our results indicate that N. oleophilus might be able to enzymatically denitrify nitrite to N2O at low pH.