Long-term diversified crop rotations alter the soil organic carbon (SOC) content. However, the mechanisms underlying C fraction regulation by soil functional genes and associated microbial communities remain poorly understood. The present study explored the effects of long-term crop rotation on soil C fraction content, soil microbial C-degradation genes, and associated microbial communities in the Loess Plateau. Compared to continuous winter wheat, corn-wheat-millet and alfalfa-potato-wheat rotations significantly increased SOC content by 31.1 % and 47.8 %, respectively. Moreover, these crop rotation regimes led to different degrees of enhancement in soil C fractions, including particulate organic C, potential C mineralization and microbial biomass C. Compared to bare land, continuous winter wheat increased the abundance of C-degradation functional genes by 11.4 %, whereas corn-wheat-millet rotation enhanced it by 15.7 %, pea-wheat-millet rotation by 18.2 %, and alfalfa-potato-wheat rotation by 19.1 %. Bulk density and soil total nitrogen were significantly correlated with soil microbial C-degradation genes and associated microbial communities. The abundance of soil C-degradation genes and microbial diversity was positively correlated with soil C fractions. Moreover, functional genes responsible for lignin and cellulose degradation, including ligB and bglX, were significantly positively correlated with the soil C fraction content. Our findings indicate that long-term diversified crop rotations, such as corn-wheat-millet or alfalfa-potato-wheat, enhance soil C sequestration and soil quality, and provide practical guidance for sustainable agricultural management.