Intensive crop production on grassland-derived Mollisols has liberated massive amounts of carbon (C) to the atmosphere. Whether minimizing soil disturbance, diversifying crop rotations, or re-establishing perennial grasslands and integrating livestock can slow or reverse this trend remains highly uncertain. We investigated how these management practices affected soil organic carbon (SOC) accrual and distribution between particulate (POM) and mineral-associated (MAOM) organic matter in a 29-y-old field experiment in the North Central United States and assessed how soil microbial traits were related to these changes. Compared to conventional continuous maize monocropping with annual tillage, systems with reduced tillage, diversified crop rotations with cover crops and legumes, or manure addition did not increase total SOC storage or MAOM-C, whereas perennial pastures managed with rotational grazing accumulated more SOC and MAOM-C (18 to 29% higher) than all annual cropping systems after 29 y of management. These results align with a meta-analysis of data from published studies comparing the efficacy of soil health management practices in annual cropping systems on Mollisols worldwide. Incorporating legumes and manure into annual cropping systems enhanced POM-C, microbial biomass, and microbial C-use efficiency but did not significantly increase microbial necromass accumulation, MAOM-C, or total SOC storage. Diverse, rotationally grazed pasture management has the potential to increase persistent soil C on Mollisols, highlighting the key role of well-managed grasslands in climate-smart agriculture.