Long term fertilization may have a significant effect on soil organic carbon (SOC) fractions and profile distribution. However, previous research mostly explored the SOC in the topsoil and provided little or no information about its distribution in deeper layers and various protection mechanisms particularly under long-term fertilization. The present study investigated the contents and profile distribution (0–100 cm) of distinct SOC protection mechanisms in the Mollisol (black soil) of Northeast China after 35 years of mineral and manure application. The initial Organic Matter content of the topsoil (0–20 cm) ranged from 26.4 to 27.0 g kg−1 soil, and ploughing depth was up to 20 cm. A combination of physical-chemical fractionation methods was employed to study various SOC fractions. There were significant variations throughout the profile among the various fractions and protection mechanisms. In topsoil (to 40 cm), mineral plus manure fertilization (MNPK) increased the total SOC content and accounted for 16.15% in the 0–20 cm and 12.34% in the 20–40 cm layer, while the manure alone (M) increased the total SOC by 56.14%, 48.73% and 27.73% in the subsoil (40–60, 60–80 and 80–100 cm, respectively). Moreover, MNPK and M in the topsoil and subsoil, respectively increased the unprotected coarse particulate organic carbon (cPOC) (48% and 26%, respectively), physically protected micro-aggregate (μagg) (20% and 18%, respectively) and occluded particulate organic carbon (iPOC) contents (279% and 93%, respectively) compared with the control (CK). A positive linear correlation was observed between total SOC and the cPOC, iPOC, physico-biochemically protected NH-μSilt and physico-chemically protected H-μSilt (p < 0.01) across the whole profile. Overall, physical, physico-biochemical and physico-chemical protection were the predominant mechanisms to sequester carbon in the whole profile, whereas the biochemical protection mechanisms were only relevant in the topsoil, thus demonstrating the differential mechanistic sensitivity of fractions for organic carbon cycling across the profile.