AbstractSoil organic carbon (SOC) transformation is susceptible to tillage practices. Conservation tillage is known to optimize soil structure, improve microbial community diversity and increase SOC storage. However, how soil aggregate distribution and microbial community structure and function within aggregates affect SOC transformation under long‐term conservation tillage remains unclear. In this study, SOC mineralization dynamics were studied in situ and under laboratory conditions to examine the mechanisms by which C functional genes within soil aggregates of different sizes (i.e., mega‐, macro‐, and micro‐aggregates) influence SOC mineralization under long‐term tillage (i.e., zero, chisel, and plow tillage) in a dryland. The results indicated that in the winter wheat and summer maize rotation cropping system, SOC‐derived CO2‐C emissions were 143.99 and 133.29 g CO2‐C m−2 h−1 lower under chisel and zero tillage than that under plow tillage, respectively. Moreover, after 180 days of laboratory incubation, SOC mineralization in micro‐ and macro‐aggregates was 1.98 and 1.63 mg CO2‐C g−1 d−1 higher than that in mega‐aggregates, respectively. The aggregate‐associated differential modules of bacterial co‐occurring networks may be directly governed by bacterial community diversity and composition, which might play critical roles in driving SOC mineralization in response to different tillage intensities. Moreover, aggregate‐associated functional genes involved in labile and recalcitrant C compositions, which were determined by shotgun metagenomic sequencing, were associated with SOC mineralization and were significantly affected by the legacy effect of tillage intensity and aggregate size. Particularly, partial least squares path modeling revealed that genes involved in simple sugar metabolism exerted significantly positive effects on SOC mineralization, except for the effects of tillage intensity and aggregate size. Overall, this study showed that decreased abundances of labile C decomposition‐related functional genes within aggregates and community composition changes, as elucidated by the differences in bacterial network modules, under conservation tillage inhibit SOC mineralization. These findings may help in the development of adaptive soil tillage strategies for reducing carbon emissions in agroecosystems.