Nitrous oxide efflux from soil is an important ecological process in terms of global climate impacts, stratospheric chemistry, and soil fertility. The effects of grassland restoration on nitrous oxide (N2O) efflux in formerly cultivated agricultural soils are not well known. Restoration changes the storage and availability of soil C and N, with potential consequences for N2O efflux. We examined changes in potential N2O efflux across a 35-year chronosequence of grassland restorations, using lab incubations at moisture levels that maximized N2O emissions, to quantify the relationship between N2O efflux and soil properties known to change predictably during grassland restoration. We found that restoring cultivated agricultural land to grassland rapidly decreased N2O efflux from soils, though native prairie had a potential N2O efflux higher than the agricultural land. The oldest restoration had N2O efflux 50 times lower than native prairie. Changes in N2O efflux were more strongly correlated with N mineralization than C mineralization, according to multiple regression analysis. These results suggest that grassland restoration could mitigate nitrous oxide emissions for decades.