Many croplands planted to perennial grasses under the Conservation Reserve Program are being returned to crop production, and with potential consequences for water quality. The objective of this study was to quantify the impact of grassland-to-cropland conversion on nitrate-nitrogen (NO₃–N) concentrations in soil and shallow groundwater and to assess the potential for perennial filter strips (PFS) to mitigate increases in NO₃–N levels. The study, conducted at the Neal Smith National Wildlife Refuge (NSNWR) in central Iowa, consisted of a balanced incomplete block design with 12 watersheds and four watershed-scale treatments having different proportions and topographic positions of PFS planted in native prairie grasses: 100% rowcrop, 10% PFS (toeslope position), 10% PFS (distributed on toe and as contour strips), and 20% PFS (distributed on toe and as contour strips). All treatments were established in fall 2006 on watersheds that were under bromegrass (Bromus L.) cover for at least 10 yr. Nonperennial areas were maintained under a no-till 2-yr corn (Zea mays L.)–soybean [Glycine max (L.) Merr.] rotation since spring 2007. Suction lysimeter and shallow groundwater wells located at upslope and toeslope positions were sampled monthly during the growing season to determine NO₃–N concentration from 2005 to 2008. The results indicated significant increases in NO₃–N concentration in soil and groundwater following grassland-to-cropland conversion. Nitrate-nitrogen levels in the vadose zone and groundwater under PFS were lower compared with 100% cropland, with the most significant differences occurring at the toeslope position. During the years following conversion, PFS mitigated increases in subsurface nitrate, but long-term monitoring is needed to observe and understand the full response to land-use conversion.