In winter wheat systems, crop nitrogen(N) uptake is sourced primarily from inorganic N fertilization and N mineralization from soil organic matter(SOM). N mineralization is a dynamic N cycling process that relies on many different biological and chemical interactions within the soil. However, N fertilization potentially disrupts this process via changes in soil biological functioning. While N fertilization can increase total N in soil, the storage and availability of increased total N within SOM are unclear. Additionally, research has shown a direct suppression of N mineralization because of fertilization. The goal of this study was to understand how N fertilization alters the availability of N to be mineralized in soil and affects microbial groups and functioning related to N mineralization. Archived samples from a long-term NPK rate trial in a winter wheat system were used to evaluate changes in N storage between two distinct SOM fractions. Total N of both fractions increased with N fertilization rate, but a greater distribution of total N accumulated within available fractions as N fertilization increased. Fresh soils from the trial were sampled to measure a suite of microbial measurements including phospholipid fatty acid analysis(PLFA), CO2 respiration, microbial biomass, and gross N mineralization rates. Results showed significant differences and trends among treatments. PLFA revealed decreased abundances of gram+/gram- ratios, fungi/bacteria ratios, and arbuscular mycorrhizal fungi. Microbial biomass C and N showed initial decreases in response to N fertilization, but then increased at higher rates. Data showed a potential for increased N availability as a result of fertilization, but decreases in important microbial groups and functions that may be involved in N mineralization.