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The effects of long-term film mulching and primary nitrogen (N) fertilization strategies on the microbial processes underlying N2O emissions from calcareous upland soils are poorly understood. A two-year field experiment including five treatments viz. no mulching with recommended N (N225), film mulching with recommended N (F225), high dose N (F380), manure addition (F225+M), and no N (F0) was conducted to explore the N2O production pathways in a maize (Zea mays L.) cultivation system using combined isotopocule and quantitative PCR analysis. The cumulative N2O emissions were 0.56 − 0.68, 0.70 − 0.71, 1.06 − 1.07, 1.50 − 1.54, and 0.16 − 0.19 kg N2O-N ha−1 in N225, F225, F380, F225+M and F0 treatments, respectively. Nitrifier denitrification and/or classical denitrification accounted for 53.4 − 85.0% of the total N2O emissions in the high N2O emission period. Long-term film mulching changed the N2O-related community composition and led to a significantly higher fraction of bacterial denitrification and/or nitrifier denitrification (fdenitrification) in most events, compared with those in bare land. A relatively low fdenitrification and high N2O/(N2O + N2) ratio often occurred in the F225+M (in mixing-reduction scenario) and F380 treatments. Correlation analysis suggested that N2O fluxes were positively correlated with NO3−-N, dissolved organic N, total N, soil temperature, soil organic carbon, and the abundance of AOB amoA and nosZ genes, whereas they were negatively correlated with soil pH and C/N. Overall, our results suggest that long-term film mulching and N fertlization strategies could impact the processes of N2O production and consumption, whereas denitrification was still the primary N2O production pathways. [ABSTRACT FROM AUTHOR]