Here, we examine the effect of long-term pH differences and short-term pH change on N₂O emissions from soil, and the microbial source (ammonia oxidation versus denitrification) of ¹⁵N-N₂O emissions. ¹⁵N-fertiliser (20 g N m⁻²; 10 atom% excess ¹⁵N) was applied to (1) a silt loam soil of pH 7 held at 50% and 65% water-filled pore space (WFPS) (experiment 1) and (2) a loamy sand soil maintained at pH 4.5 and pH 7 for over 40 years (experiment 2). Soils were limed with CaCO₃ or acidified with H₂SO₄, and comparisons were made with unadjusted soils. Ammonia oxidation was the main microbial source of ¹⁵N-N₂O in soils limed to pH 7.0-8.1, unadjusted pH 7.1 (Experiment 1) and long-term pH 7 (experiment 2) soils. Eighty percent of ¹⁵N-N₂O from the long-term pH 4.5 soil (experiment 2) was derived from denitrification suggesting a possible inhibition of N₂O reduction. Short-term acidification to pH 5.6 or 4.3 lowered N₂O emissions. Liming of the pH 4.5 soil resulted in over four times greater N₂O emission (11 mg ¹⁴⁺¹⁵N-N₂O m⁻² over 41 days) than from the long-term pH 7.0 soil (experiment 2), with an associated increase in ammonia oxidiser-N₂O and decrease in denitrifier-N₂O production. This is the first report of a pH-induced change in microbial source of N₂O. Our results highlight the importance of distinguishing between short- and long-term effects of pH management when predicting N₂O emissions from soil, as they exhibit predominance of different microbial groups in N₂O production, with likely adaptation of the microbial community.