The fate of soil organic carbon (SOC) sequestered by afforestation is crucial for the mitigation of the anthropogenic climate change but remains largely unclear. This lack of knowledge is particularly true for SOC turnover driven by enzyme activity. Here we measured hydrolase (including β-glucosidase, α-glucosidase, cellobiohydrolase and xylanase) and oxidase (including polyphenol oxidase and peroxidase) activities in soil aggregates following 30-year afforestation in central China. We also analyzed the relationships of enzyme activities with SOC concentrations, soil C:nitrogen (N) ratios and δ 13 C values of soil organic pool (removing any carbonates by acid hydrolysis) and stable pool (NaOCl–resistant). Afforestation significantly enhanced soil β-glucosidase, α-glucosidase and xylanase activities in bulk soil, as well as SOC concentrations in bulk soil and all aggregate fractions compared to those in the open area and cropland. In particular, the woodland increased SOC concentration in >2000 μm macroaggregates by 4.2- and 3.2-fold, compared to the open area and cropland, respectively. Soil hydrolase activities were generally lower but SOC concentrations were higher in >2000 μm macroaggregates compared with those in other aggregate fractions following afforestation. Hydrolase activities were negatively correlated with SOC and C:N ratios in soil aggregate fractions following afforestation. Results of structural equation modeling indicated that the increasingly inhibited hydrolase activities with increasing soil aggregate size indirectly promoted SOC sequestration following afforestation. In addition, both hydrolase and oxidase were positively correlated with δ 13 C values in the stable pool of the afforested soils, confirming the essential role of enzymes for SOC turnover in soil aggregates following afforestation. Overall, our results highlight the importance of unevenly distributed enzyme activities among soil aggregates in regulating SOC sequestration following afforestation.