Increasing the recalcitrant C pool in soils requires the expansion of sorptive surface areas that promote organo-mineral complexes within soil aggregates. Additional connectivity of intra-aggregate pore networks connectivity, formed during repeated soil drying and rewetting (DW), provides highly functional pathways for the diffusion of dissolved organic C (DOC) compounds onto new sorptive surface areas. We investigated the spatial distribution and dynamics of ¹³C-glucose supplied to individual aggregates, spatial differentiation of microbial communities within aggregates, and their contribution to aggregate stabilization during incubations associated with multiple DW cycles. DW cycles with no glucose additions significantly reduced aggregate stability and microbial community diversity with no gradient between exterior and interior layers. Additions of glucose-¹³C with each DW cycle (total 1250 μg C) maintained soil aggregate stability throughout the 35-day incubation period and led to spatial gradients of ¹³C content within aggregates. Rapid decreases in the mineralization rate of glucose-¹³C during incubation indicated either physical protection or chemical stabilization of newly added ¹³C within aggregate interiors. DW cycles containing additions of glucose increased the diversity of microbial communities within aggregates, with greatest increases in the exterior layers of soil macroaggregates.