Denitrification is reducing nitrate to nitrous oxide (N2O) and nitrogen (N2) under the action of denitrifying microorganisms, which are the main source of soil N2O emissions. However, the co-occurrence pattern and community assembly process of denitrifier communities have not been studied in agricultural soils with long-term contrasting tillage histories. Based on a continuous (> 11 yr) conservation experiment, we aimed to understand the community assembly process and co-occurrence network of nirK-, nirS-, nosZI- and nosZII-type denitrifier communities under conservation tillage (i.e., zero tillage (ZT) and chisel plough tillage (CPT)) and conventional tillage (i.e., plow tillage (PT)). Continuous in situ measurements showed that long-term conservation tillage significantly decreased the denitrification potential and N2O emission fluxes. CPT and ZT significantly increased the taxonomic and phylogenetic diversity of nirK, nosZI, and nosZII type communities, whereas consistently decreased the diversity of nirS-type communities, compared with PT. Although the assembly of nirK, nirS, nosZI, and nosZII type denitrifier communities were dominated by stochastic processes, the relative importance of stochastic assembly of denitrifying microbial communities was higher in conservation tillage soil. The co-occurrence network analyses further revealed clear tillage-induced ecological functions of N2O emissions, as evidenced by lower key nodes abundance and robustness of nirS and nirK communities network and higher key nodes abundance and robustness of nosZI and nosZII communities in conservation tillage soils. Variation partitioning analysis further indicated that the assembly processes significantly changed soil N2O emission intensity by regulating α-diversity and the key species abundances in the co-occurrence network of denitrifying communities. Overall, conservation tillage practices increased the diversity and the key nodes abundance of nosZI- and nosZII-type communities and decreased the diversity and the key nodes abundance of nirS- and nirK-type communities by regulating the relative importance of the stochastic assembly process of denitrifier communities, which is eventually likely to curb N2O emissions by strengthening the capacity of the N2O sink and weakening the capacity of the N2O source. These findings provide new insights into the formation of dryland microbial communities in agricultural soils with long-term contrasting tillage histories. Especially considering future climate change, this knowledge would prove useful in improving agroecosystem productivity and curbing greenhouse gas emissions.