Soil organic carbon (SOC), a core soil quality indicator, is influenced by management practices. The objective of our 2012-2016 study was to elucidate the impact of gypsum, crop rotation, and cover crop on SOC and several of its biological indicators under no-till in Alabama (Shorter), Indiana (Farmland), and Ohio (Hoytville and Piketon) in the USA. A randomized complete block design in factorial arrangement with gypsum (at 0, 1.1, and 2.2 Mg/ha annually), rye (Secale cereal L.) vs no cover crop, and rotation (continuous soybean [Glycine max (L) Merr., SS] vs corn [Zea mays, L.]-soybean, both the CS and SC phases) was conducted. Composite soils were collected (0-15 cm and 15-30 cm) in 2016 to analyze microbial biomass C (SMBC), SOC, total N, active C, cold and hot-water extractable C, C and N pool indices (CPI and NPI), and C management index (CMI). Results varied for main effects of gypsum, crop rotation, and cover crop on SOC pools, total N, and SOC lability within and across the sites. Gypsum at 2.2 Mg/ha increased SMBC within sites and by 41% averaged across sites. Likewise, gypsum increased SMBC:SOC, active C, and hot-water C (as indicators of labile SOC) averaged across sites. CS rotation increased SOC, active C, CPI, and CMI compared to SS, but decreased SMBC and SMBC:SOC within and across sites. CPI had a significant relationship with NPI across all sites (R2 = 0.90). Management sensitive SOC pools that responded to the combined gypsum (2.2 Mg/ha), crop rotation (CS), and cover crop (rye) were SMBC, SMBC:SOC, active C, and CMI via SMBC. These variables can provide an early indication of management-induced changes in SOC storage and its lability. Our results show that when SOC accumulates, its lability has decreased, presumably because the SMBC has processed all readily available C into a less labile form.