Aims <p>Maintaining food production while reducing nitrogen loss is the main challenge for agricultural intensification. For nitrate transformation pathways, increasing the proportion of the dissimilated nitrate nitrogen reduction (DNRA) process contributes to nitrogen conservation. Intercropping as an effective cropping system, can promote the sustainability of agriculture. However, the mechanism of how intercropping and fertilization interactions affect the nitrogen transformation processes in agricultural soils remains largely unresolved.</p> Methods <p>¹⁵N-isotope tracing combined with quantitative PCR and metagenomics sequencing were used to determine nitrogen conversion rates and gene abundance in the soybean field of southwest China, which has been treated with two cropping systems and five fertilization treatments.</p> Results <p>We found that cropping systems interact with fertilization co-shaped microbial nitrogen cycling. Soybean-maize intercropping significantly increased DNRA rates by 105% with the low organic fertilization. Moreover, DNRA <i>nrfA</i> (by 346%) gene abundance were increased in the intercropping than monoculture under the low organic fertilization. Random forest analysis showed that the DNRA process was mainly regulated by soil NO₃⁻ concentration (9.3%) and <i>nrfA</i> gene (11.2%). Furthermore, 21 high qualities of metagenome-assembled genomes (MAGs) were categorized to be involved in nitrogen-cycling pathways. MAG115, MAG12, and MAG91 were identified as the key MAGs and they were taxonomically classified as <i>Pedosphaerales</i>, <i>Streptomycetaceae</i>, <i>Burkholderiales</i> for driving DNRA process.</p> Conclusions <p>Collectively, our results imply that intercropping with low organic fertilizers input could promotes DNRA rates for nitrogen sustainability, and these findings lay the foundation for developing reasonable nitrogen fertilizer management and cropping systems under agricultural intensification.</p> Graphical Abstract <p></p>