Soil C change and CO2 emission due to different tillage systems need to be evaluated to encourage the adoption of conservation practices to sustain soil productivity and protect the environment. We hypothesize that soil C storage and CO2 emission respond to conservation tillage differently from conventional tillage because of their differential effects on soil properties. This study was conducted from 1998 through 2001 to evaluate tillage effects on soil C storage and CO2 emission in Clarion-Nicollet-Webster soil association in a corn [Zea mays L.]-soybean [Glycine max (L.) Merr.] rotation in Iowa. Treatments included no-tillage with and without residue, strip-tillage, deep rip, chisel plow, and moldboard plow. No-tillage with residue and strip-tillage significantly increased total soil organic C (TC) and mineral fraction C (MFC) at the 0- to 5- and 5- to 10-cm soil depths compared with chisel plow after 3 yr of tillage practices. Soil CO2 emission was lower for less intensive tillage treatments compared with moldboard plow, with the greatest differences occurring immediately after tillage operations. Cumulative soil CO2 emission was 19 to 41% lower for less intensive tillage treatments than moldboard plow, and it was 24% less for no-tillage with residue than without residue during the 480-h measurement period. Estimated soil mineralizable C pool was reduced by 22 to 66% with less intensive tillage treatments compared with moldboard plow. Adopting less intensive tillage systems such as no-tillage, strip-tillage, deep rip, and chisel plow and better crop residue cover are effective in reducing CO2 emission and thus improving soil C sequestration in a corn-soybean rotation.