Background and aims <p>Differences in farming practices play a crucial role in shaping farmland sustainability, soil quality, and global warming potential (GWP). While no-tillage (NT) practices have been extensively studied in widely adopted cropping systems such as wheat-corn and rice monoculture, the effects of no-tillage on oilseed rape-rice rotation systems remain underexplored. In particular, the microbial-driven mechanisms involved remain poorly understood.</p> Methods <p>The study was conducted over a 2-year cycle of an oilseed rape-rice rotation system. Soil greenhouse gas emissions and carbon sequestration under NT and CT management practices were measured and compared. Additionally, high-throughput sequencing of soil methanogens and methane-oxidizing bacteria was performed.</p> Results <p>Compared with CT, NT significantly reduced CH₄ and CO₂ emissions by 20.3% and 16.6%, respectively. Although N₂O emissions increased by 24.0%, the overall GWP decreased by 9.9%. NT also increased the carbon efficiency ratio (CER, the ratio of grain yield (carbon content) to carbon equivalent emissions) by 26.6% and increased soil organic carbon by 8.6% over the 2-year period. High-throughput sequencing revealed that NT significantly enhanced the diversity and abundance of methane-oxidizing microorganisms (methanotrophs, which mitigate methane by converting it to CO₂ under aerobic conditions) and altered the relative abundance of dominant genera compared to CT.</p> Conclusion <p>NT is an effective strategy to reduce CH₄ emissions, enhance soil and crop carbon fixation, and increase the diversity of soil methane-oxidizing bacteria in oilseed rape-rice rotation. This practice offers substantial environmental benefits, contributing to sustainable farmland development and mitigating global warming potential.</p>