Empirical studies show that nitrogen (N) addition often reduces microbial decomposition of soil organic matter (SOM) and carbon dioxide (CO2) production via microbial respiration. Although predictions from theoretical models support these findings, the mechanisms that drive this response remain unclear. To address this uncertainty, we sampled soils of three grassland sites in the U.S. Central Great Plains that each have received seven years of continuous experimental nutrient addition in the field. Nitrogen addition significantly decreased the decomposition rate of slowly cycling SOM and the cumulative carbon (C) respired per mass soil C. We evaluated whether this effect of N addition on microbial respiration resulted from: 1) increased microbial carbon use efficiency (CUE), 2) decreased microbial oxidative enzyme activity, or 3) decreased microbial biomass due to plant and/or soil mediated responses to N enrichment. In contrast to our hypotheses – as well as results from N addition studies in forest ecosystems and theoretical predictions – N did not increase microbial CUE or decrease microbial oxidative enzyme activity. Instead, reduced microbial biomass likely caused the decreased respiration in response to N enrichment. Identifying what factors drive this decreased microbial biomass response to N should be a priority for further inquiry.