Conservation tillage has been shown to mitigate climate change by promoting the sequestration of soil carbon (C) in agroecosystems. However, knowledge on how conservation tillage accumulates soil organic C (SOC), especially at the aggregate scale, remains limited. This study aimed to clarify the effects of conservation tillage on SOC accumulation by measuring hydrolytic and oxidative enzyme activities and C mineralization in aggregates and developing an extended scheme of C flows between aggregate fractions using the 13C natural abundance (δ13C) method. Topsoils (0–10 cm) were sampled from a 21-year tillage experiment located in the Loess Plateau of China. Compared with conventional (CT) and reduced tillage with straw removal (RT), no-till (NT) and subsoiling with straw mulching (SS) enhanced the proportions of macro-aggregates (> 0.25 mm) (by 12–26%) and SOC contents in bulk soils and all aggregate fractions (by 12–53%). In bulk soils and all aggregate fractions, SOC mineralization and the activities of hydrolases (β-1,4-glucosidase, β-acetylglucosaminidase, β-xylosidase, and cellobiohydrolase) and oxidases (peroxidase and phenol oxidase) were 9–35% and 8–56% lower, respectively, under NT and SS than under CT and RT. Partial least squares path model revealed that reductions in the activities of hydrolases and oxidases and increases in macro-aggregation decreased SOC mineralization in bulk soils and macro-aggregates. Furthermore, Δ13C values (aggregate-associated δ13C − bulk-soil δ13C) increased with decreasing size of soil aggregates, suggesting that C is younger in larger aggregates than in smaller aggregates. The probability of C flows from large to small soil aggregates was lower under NT and SS than under CT and RT, indicating that young SOC with low rates of decomposition in macro-aggregates was better protected under NT and SS. Overall, NT and SS enhanced SOC accumulation in macro-aggregates by decreasing the activities of hydrolases and oxidases and C flows from macro- to micro-aggregates, which promoted C sequestration in soils. The present study provides improved insights into the mechanism and prediction of soil C accumulation under conservation tillage.