Isotopocule signatures of N2O (δ15Nbulk, δ18O and site preference) are useful for discerning soil N2O source, but sometimes, N fertilizer is needed to ensure that there is enough N2O flux for accurate isotopocule measurements. However, whether fertilizer affects these measurements is unknown. This study evaluated a gradient of NH4NO3 addition on N2O productions and isotopocule values in two acidic subtropical soils. The results showed that N2O production rates obviously amplified with increasing NH4NO3 (p < 0.01), although a lower N2O production rate and an increasing extent appeared in forest soil. The δ15Nbulk of N2O produced in forest soil was progressively enriched when more NH4NO3 was added, while becoming more depleted of agricultural soil. Moreover, the N2O site preference (SP) values collectively elevated with increasing NH4NO3 in both soils, indicating that N2O contributions changed. The increased N2O production in agricultural soil was predominantly due to the added NH4NO3 via autotrophic nitrification and fungal denitrification (beyond 50%), which significantly increased with added NH4NO3, whereas soil organic nitrogen contributed most to N2O production in forest soil, probably via heterotrophic nitrification. Lacking the characteristic SP of heterotrophic nitrification, its N2O contribution change cannot be accurately identified yet. Overall, N fertilizer should be applied strictly according to the field application rate or N deposition amount when using isotopocule signatures to estimate soil N2O processes.