Under semiarid subtropical field conditions, denitrification was measured from the arable soil layer of an irrigated wheat–maize cropping system fertilized with urea at 50 or 100 kg N ha−1 year−1 (U50 and U100, respectively), each applied in combination with 8 or 16 t ha−1 year−1 of farmyard manure (FYM) (F8 and F16, respectively). Denitrification was measured by acetylene inhibition/soil core incubation method, also taking into account the N2O entrapped in soil cores. Denitrification loss ranged from 3.7 to 5.7 kg N ha−1 during the growing season of wheat (150 days) and from 14.0 to 30.3 kg N ha−1 during the maize season (60 days). Most (up to 61%) of the loss occurred in a relatively short spell, after the presowing irrigation to maize, when the soil temperature was high and a considerable NO3 −-N had accumulated during the preceding 4-month fallow; during this irrigation cycle, the lowest denitrification rate was observed in the treatment receiving highest N input (U100+F16), mainly because of the lowest soil respiration rate. Data on soil respiration and denitrification potential revealed that by increasing the mineral N application rate, the organic matter decomposition was accelerated during the wheat-growing season, leaving a lower amount of available C during the following maize season. Denitrification was affected by soil moisture and by soil temperature, the influence of which was either direct, or indirect by controlling the NO3 − availability and aerobic soil respiration. Results indicated a substantial denitrification loss from the irrigated wheat–maize cropping system under semiarid subtropical conditions, signifying the need of appropriate fertilizer management practices to reduce this loss.