© 2015 Elsevier Ltd. Understanding the influence of long-term crop management practices on the soil microbial community is critical for linking soil microbial flora with ecosystem processes such as those involved in soil carbon cycling. In this study, pyrosequencing and a functional gene array (GeoChip 4.0) were used to investigate the shifts in microbial composition and functional gene structure in a medium clay soil subjected to various cropping regimes. Pyrosequencing analysis showed that the community structure (β-diversity) for bacteria and fungi was significantly impacted among different cropping treatments. Functional gene array-based analysis revealed that crop rotation practices changed the structure and abundance of genes involved in C degradation. Significant correlations were observed between the activities of four enzymes involved in soil C degradation and the abundance of genes responsible for the production of respective enzymes, suggesting that a shift in the microbial community may influence soil C dynamics. We further integrated physical, chemical, and molecular techniques (qPCR) to assess relationships between soil C, microbial derived enzymes and soil bacterial community structure at the soil micro-environmental scale (e.g. within different aggregate-size fractions). We observed a dominance of different bacterial phyla within soil microenvironments which was correlated with the amount of C in the soil aggregates suggesting that each aggregate represents a different ecological niche for microbial colonization. Significant effects of aggregate size were found for the activity of enzymes involved in C degradation suggesting that aggregate size distribution influenced C availability. The influence of cropping regimes on microbial and soil C responses declined with decreasing size of soil aggregates and especially with silt and clay micro-aggregates. Our results suggest that long term crop management practices influence the structural and functional potential of soil microbial communities and the impact of crop rotations on soil C turnover varies between different sized soil aggregates. These findings provide a strong framework to determine the impact of management practices on soil C and soil health.