Nitrogen (N) losses from intensified agriculture are a major cause of global change, due to nitrate (NO3-) export and the eutrophication of aquatic systems as well as emissions of nitrous oxide (N2O) into the atmosphere. Diversified agroecosystems with legume cover crops couple N and carbon (C) inputs to soil and reduce N pollution, but there is a need to identify controls on legume N2 fixation across ecosystems with variable soil conditions. Here, I tested the hypothesis that N mineralization from turnover of soil organic matter (SOM) regulates legume N2 fixation across 10 farms that spanned a gradient of SOM levels. I separated soil samples into two SOM fractions, based on size and density, which are indicators of soil nutrient cycling and N availability (free particulate organic matter and intra-aggregate particulate organic matter (POM)). This study indicates downregulation of legume N2 fixation in diversified agroecosystems with increasing N availability in intra-aggregate POM and increasing N mineralization. Intercropping the legume with a grass weakened the relationship between N in POM and N2 fixation due to N assimilation by the grass. Further, mean rates of N and C mineralization across sites increased with two seasons of a legume-grass cover crop mixture, which could enhance this stabilizing feedback between soil N availability and N2 fixation over time. These results suggest a potential mechanism for the diversity-ecosystem function relationships measured in long-term studies of agroecosystems, in which regular use of legume cover crops increases total soil organic C and N and reduces the environmental costs of crop production.