Abstract The signaling compound nitric oxide (NO) might play an important, yet unquantified role in mediating soil biogeochemical Carbon and Nitrogen cycles. This study quantified the effects of different soil-typical exogenous NO concentrations on the microbial community, on fertilizer N turnover, and on C and N trace gas fluxes of agricultural soil. For this, we repeatedly established soil NO concentrations of either 0, 200, 400, and ppbv˗NO in soil mesocosms for in total of 12 days, followed by high-resolution automated measurements of CO 2 , NO, CH 4 , and N 2 O fluxes, molecular analysis of microbial community composition and 15 N-isotope-tracing based assessment of fertilizer N turnover. We found no effects of different NO levels on microbial communities and CO 2 , CH 4 , and NO fluxes. However, at 200 ppbv concentration, exogenous NO promoted microbial assimilation of fertilizer N. In contrast, at 400 ppbv˗NO concentration, microbial biomass N was reduced, and microbial uptake of fertilizer N was inhibited, accompanied by a 33% reduction of N 2 O emissions. This suggested a promoting effect of 200 ppbv˗NO on the physiology of cells involved in heterotrophic microbial N turnover, probably reinforcing the role of cell-endogenous NO. In contrast, the higher exogenous NO concentrations of 400 ppbv seemed to inhibit heterotrophic microbial inorganic N assimilation, with however no increase in N 2 O emissions due to detoxification mechanisms. In conclusion, our pioneering study provides first insights into impacts of exogenous NO on soil C and N biogeochemistry in natural soil systems and reveals a NO concentration-dependent regulation of microbial N retention.