The discovery of complete ammonia oxidizers (comammox) has dramatically altered our perception of nitrogen (N) biogeochemistry. However, their functional importance vs. the canonical ammonia oxidizers (i.e., ammonia oxidizing-archaea (AOA) and bacteria (AOB)) in agroecosystems is still poorly understood. Accordingly, a new assay, which involves the combined use of acetylene, 3,4-dimethylpyrazole phosphate (DMPP), and 1-octyne, was adopted to assess the ammonia (NH3) oxidation and nitrous oxide (N2O) production activity of these functional guilds in a subtropical Inceptisol subjected to long-term different fertilization regimes, namely CK (no fertilizer control), synthetic fertilizer only (NPK), organic manure only (M) and organic manure plus synthetic fertilizer (MNPK). AOA dominated the NH3 oxidation in M treatment, whereas AOB dominated all the NH3 oxidation and N2O production processes in all but M treatment, and comammox played a minor role in both NH3 oxidation and N2O production in all treatments. Both M and MNPK treatments significantly increased the activity and growth of comammox. Compared to NPK, comammox exhibited increases of 270 % and 326 % in the NH3 oxidation rates, and increases of 1472 % and 563 % in the N2O production rates in M and MNPK, respectively. Random forest model revealed that copper (Cu), comammox abundance, and dissolved organic nitrogen (DON) were the most important predictors for the NH3 oxidation rates of comammox. Redundancy analyses (RDA) showed that fertilizer treatments significantly altered the community composition of NH3 oxidizers, and pH was the overarching parameter underpinning the community shift of the NH3 oxidizers. Overall, this paper provides evidence that comammox play a minor yet unneglectable role in the nitrification of agroecosystems, and the long-term addition of organic manure stimulates the growth and activity of comammox in a subtropical Inceptisol.