Reductions of nitrogen (N) export from agricultural lands because of changes in specific N stocks and fluxes by incorporation of small amounts of prairie vegetation strips (PVS) are poorly understood. The primary objective of this study was to evaluate the effect of the presence and topographical position of PVS on soil and plant carbon (C) and N stocks relative to annual crop and native prairie vegetation. The study was implemented within three small adjacent watersheds, treated with one of the following cover types: (1) 100% row-crop agriculture (CROP); (2) 20% prairie vegetation (PVS) distributed along the contour across three topographical positions: upslope, sideslope and footslope position; and (3) 100% 17-year old reconstructed native prairie (RNP) as the control condition. Total soil organic C (SOC), total soil N (TN), inorganic N availability as indexed by ion exchange resins, N stocks in plant biomass and litter, and the ratio of C₃:C₄ plant species were measured during the 2010 growing season. Results showed that over five years of treatment, PVS footslope improved soil quality by increasing TN by almost 100% and SOC by 37%; while CROP footslope TN decreased by 31% and SOC decreased by 28%. Overall, N stocks in plant biomass and litter were higher in PVS compared with RNP, except in the footslope where the lower N plant stocks was associated with higher C₃ abundance in RNP. Nitrogen availability was higher in CROP (25.4 ± 1.4), followed by PVS (10.2 ± 1.3), and RNP (2.2 ± 1.4); with the highest values recorded in the upslope position for PVS and RNP, and the footslope for CROP. These findings are important for designing watersheds with PVS to reduce N accumulation in the footslope position and promote additional N retention in soil organic matter and plant biomass, thereby minimizing N losses to streams.