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No-tillage (NT) management is considered a leading approach for sustaining crop production and improving soil and environmental quality. Based on a long-term no-tillage experiment in a rice–oilseed rape cropping system, we examined differences in soil organic matter (SOM), soil microbial carbon (C) and nitrogen (N) content, and methane (CH₄) and carbon dioxide (CO₂) fluxes between NT and conventional tillage (CT) management. SOM under NT was 21.0 g kg^–1, and a significant difference was detected between 2004 and 2016. SOM increased under NT and CT by averages of 0.60 and 0.32 g kg^–1 year^–1, respectively. Soil microbial C and N content were higher under CT than under NT. However, soil C:N ratios under NT were 17.4 and 9.7% higher than the CT, respectively, whereas soil microbial C:N ratios under NT were on average 9.47 and 9.70% higher. In addition, about 70% of CO₂ net uptake and over 99% of net CH₄ emissions occurred during the rice season in May–September in the rice–oilseed rape cropping system. Annual cumulative CH₄ and daytime net ecosystem CO₂ exchange (NEE) under NT was 1813.9 g CO₂ equiv. m^–2, 10.8% higher than that under CT. Our results suggest that a higher soil microbial C:N ratio and NEE (CH₄ and daytime CO₂) could contribute to increasing SOM/C in the surface soil under NT management.