AimsCrop rotation, especially with legumes, is believed to boost soil organic carbon (SOC). However, studies in Northeast China indicated long-term maize-soybean rotation doesn't improve SOC more than maize and soybean monocropped. Further study is needed to explore why rotation system don't significantly increase SOC under conventional management.MethodsSamples were collected from a 30-year field experiment to assess soil amino sugars for quantifying microbial necromass carbon (MNC), employ pyrolysis-gas chromatography mass spectrometry (Py-GC/MS) for detailed analysis of soil organic matter (SOM) chemical composition, and utilize high-throughput sequencing to depict soil microbial community structure and functions.ResultsMaize monocropped has the highest SOC, followed by maize-soybean rotation, and soybean monocropped is the lowest. The lower SOC content in the maize-soybean rotation system, compared to maize monocropped, could be attributed to specific factors: (i) increased Ascomycetes phylum abundance affecting SOC degradation; (ii) negative correlation between Mortierellomycota phylum and MNC; (iii) elevated N-acetyl-glucosaminidase activity, indicating increased SOC decomposition. Despite lower SOC, the maize-soybean rotation system showed enhanced SOM stability with: (i) a higher fungal-derived carbon (C) to SOC ratio, indicating effective fungal-derived C enrichment; and (ii) Py-GC/MS analysis revealing greater abundance of stable SOM compounds like nitrogen-containing compounds and aromatic hydrocarbons than maize and soybean monocropped.ConclusionThis study demonstrated that the maize-soybean rotation system, by altering soil microbial communities, intensifies the decomposition of SOC, resulting in lower SOC content than continuous maize monocropped. However, the remaining SOM in this system exhibits greater stability, which is more conducive to long-term C sequestration.