Aims <p>Microorganisms in intercropping systems accelerate nitrogen (N) cycling in soil and supply N for crops compared to monoculture. However, whether fungi increase the organic N mineralization under intercropping systems compared to soils with sole crops remains unknown. We hypothesized that the fungal-mediated N mineralization in intercropping under low N conditions increases soil N mineralization because fungi decompose complex organic substances by secreting extracellular enzymes.</p> Methods <p>Soil was sampled in a 13-year field experiment with monocultures of maize and soybean as well as maize/soybean intercropping without N input (N0) or with 180&#xa0;kg N ha⁻¹ input (N180). The net N mineralization and the enzymes (urease and protease) as well as fungal community structure were analyzed in soil during vegetation season to assess the role of fungi in N mineralization in intercropping systems.</p> Results <p>Intercropping increased the net N mineralization in soil by 1.6 times compared to monocultured maize while the fungi did not correlate with N mineralization under N0. The redundancy analysis showed that the soil chemical properties and enzyme activities explained 68% of the total variation of fungal communities. The net mineralized N amount increased with the relative abundance of Glomeromycota under N180 (<i>p</i> = 0.04).</p> Conclusion <p>Maize/soybean intercropping exploited more N from the soil organic matter pool under&#xa0;low soil N conditions&#xa0;without the role of soil fungal communities, but the activities of phylum Glomeromycota can increase net N mineralization under high soil&#xa0;N conditions. This study provides evidence of N mineralization driven by fungi in intercropping at high N input.</p>