The immediate effects of tillage on protected soil C and N pools and on trace gas emissions from soils at precultivation levels of native C remain largely unknown. We measured the response to cultivation of CO2 and N2O emissions and associated environmental factors in a previously uncultivated U.S. Midwest Alfisol with C concentrations that were indistinguishable from those in adjacent late successional forests on the same soil type (3.2%). Within 2 days of initial cultivation in 2002, tillage significantly (P=0.001, n=4) increased CO2 fluxes from 91 to 196 mg CO2-C m−2 h−1 and within the first 30 days higher fluxes because of cultivation were responsible for losses of 85 g CO2-C m−2. Additional daily C losses were sustained during a second and third year of cultivation of the same plots at rates of 1.9 and 1.0 g C m−2 day−1, respectively. Associated with the CO2 responses were increased soil temperature, substantially reduced soil aggregate size (mean weight diameter decreased 35% within 60 days), and a reduction in the proportion of intraaggregate, physically protected light fraction organic matter. Nitrous oxide fluxes in cultivated plots increased 7.7-fold in 2002, 3.1-fold in 2003, and 6.7-fold in 2004 and were associated with increased soil NO3− concentrations, which approached 15 μg N g−1. Decreased plant N uptake immediately after tillage, plus increased mineralization rates and fivefold greater nitrifier enzyme activity, likely contributed to increased NO3− concentrations. Our results demonstrate that initial cultivation of a soil at precultivation levels of native soil C immediately destabilizes physical and microbial processes related to C and N retention in soils and accelerates trace gas fluxes. Policies designed to promote long-term C sequestration may thus need to protect soils from even occasional cultivation in order to preserve sequestered C.