Rivers are hotspots for methane (CH4) emissions, and aerobic methane oxidation is a crucial process in controlling emissions. The spatio-temporal heterogeneity of river environment can greatly affect the methane oxidation process. However, currently, few studies have focused on the spatio-temporal changes in activity of methane oxidation and the associated microbiome in riverine ecosystems, which hinders a comprehensive understanding the role of this process in reducing emissions of CH4. Here, we investigated the variations in methane oxidation activity and community of methanotrophs in sediment of a mountain river across different reaches and seasons. The potential methane oxidation rate ranged from 24.11 to 493.03 nmol CH4 g-1 (sediment) d-1, which was significantly greater in sediment obtained during the winter than in that obtained during the summer. Moreover, the rate in middle reaches was significantly greater than that in upper and lower reaches in summer. The abundance of pmoA gene of methanotrophs ranged from 2.45×10⁶ to 2.98×10⁷ copies g-1 (sediment), which was also significantly greater in winter than in summer and showed significant variations among reaches. Additionally, methanotrophic diversity and community composition exhibited significant variations across both reaches and seasons, and the relative abundance of Methylococcus and Methylocystis was closely associated with methane oxidation activity. Sediment NH4+ content, pH and temperature were potentially crucial factors affecting the activity or methanotrophic community. In conclusion, it is necessary to consider both temporal and spatial scales to improve our understanding of the significance and driving mechanisms of methane oxidation in controlling CH4 emissions from rivers.