AbstractThe 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 CO2and 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 CO2fluxes from 91 to 196 mg CO2‐C m−2 h−1and 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 CO2responses 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.