Rotational tillage is considered a potential option to improve soil organic carbon (SOC) stock and mitigate climate change. However, the mechanisms underlying SOC sequestration under rotational tillage remain poorly understood due to insufficient data on SOC concentration and mineralization within soil aggregates. A 12-year field experiment was conducted in Northwest China to evaluate the effects of tillage on SOC stocks, soil aggregate stability, aggregate-associated OC concentrations and mineralization. The results showed that rotational tillage had more crop residue and less soil disturbance, thus improving soil aggregate stability, aggregate-associated OC concentrations and SOC stocks. The highest MWD and SOC stocks were found in no-tillage rotated with subsoiling (NS), which were 36.0-69.7% and 16.3% higher than plowing, respectively. Macroaggregates had higher cumulative OC mineralization and lower OC mineralizability, due to physical protection. Rotational tillage treatments with higher soil aggregation contributed to decreasing OC mineralizability and increasing SOC sequestration. Meanwhile, rotational tillage decreased OC mineralization loss, mineralizability, and decomposition rate within microaggregates and silt-clay fractions. Among all treatments, NS treatment had the lowest total OC mineralization, which was lower by 5.94-27.3% than plowing at 0-40 cm depths. Considering soil structure stability, SOC mineralization and sequestration, NS treatment was a promising strategy in semiarid regions.