Increasing C sequestration through no-till (NT) can reduce agricultural CO2 emissions. However, for long-term NT, information is lacking on the effect of biophysical C pools and processes on C sequestration. Composite soil samples taken at 7.5-cm increments to a 30-cm depth from conventionally tilled (CT) and 2 (NT2), 23 (NT23), and 44 yr (NT44) of NT corn (Zea mays L.) plots in northwest Ohio were analyzed. The microbial biomass (SMB) was 13, 83, and 86% higher in NT2, NT23, and NT44, respectively, than in CT. No-till had slightly higher basal respiration rates but significantly lower specific maintenance respiration rates and SMB loss than CT. Aggregate stability in NT was 35 to 45% higher than in CT. Macroaggregate-protected C (CMaA) was 24, 80, and 92% higher in NT2, NT23, and NT44, respectively, than in CT. For all tillage treatments, these properties decreased with depth. The C sequestration rates for SMB were 22, 13, 7, and 3 kg ha−1 yr−1 at the 0- to 7.5-, 7.5- to 15-, 15- to 22.5-, and 22.5- to 30-cm depths, respectively for the first 10 yr of NT. During the same time, the CMaA sequestration rates were 92, 63, 47, and 37 kg ha−1 yr−1 at the four depths, and macroaggregate (MaA) formation rates were 3170, 350, 170, and 70 kg ha−1 yr−1, respectively However, these rates decreased over the 44 yr of NT. By 20 yr of NT, at all depths the C sequestered in SMB reached a plateau. Similarly, CMaA sequestration plateaued at the surface by 20 yr and MaA formation at the surface leveled out by 10 yr.