Agriculture accounts for a majority of global soil nitrous oxide (N2O) emissions. Increased crop diversity can change soil nitrogen (N), physicochemical properties and alter belowground microbial communities, leading to changes in potential N2O emissions when inorganic N fertilizer is applied. Nitrification and denitrification are two main N cycling pathways under investigation due to their contribution to soil N cycling and production of N2O. The goal of this study was to understand the impacts of 35 years of crop diversification in shaping diversity and the community size and activity of nitrifier and denitrifier bacteria and N2O emissions after N fertilization in corn. In 2017, after fertilizer addition, N2O emissions were continuously measured using automatic chambers from a long-term experiment of simple (corn-corn-soybean-soybean) and diverse (corn-corn-soybean-wheat underseeded with red clover cover crop) four year rotations under second year corn. Soil was collected during peak N2O emissions using high frequency temporal sampling to capture shifts in 16S rRNA, amoA, nirS, nirK, nosZ1 and nosZ2 genes and gene transcripts in soil where urea-ammonium-nitrate (UAN) was applied. An N2O emission event occurred in both rotations following UAN application but was higher in the diverse rotation, which also had persistent higher total and denitrifying (nirK and nosZ2) bacterial abundance. Bacterial amoA significantly increased in both rotations shortly after UAN addition, but gene detection decreased significantly in the simple rotation and remained elevated in the diverse. Transcripts for atypical nosZ2 were consistently detected but higher after UAN addition. Total bacterial diversity did not differ between simple and diverse rotations, however, abundance of microbial pathways leading to soil N2O emissions, ammonia oxidizers and denitrifiers, were elevated in soil with a more diverse cropping history. Best management practices (BMPs) that include crop diversification need to consider microbial communities and greenhouse gas (GHG) production, in order to fully quantify the soil ecosystem services.