Nitrous oxide (N 2 O), a potent greenhouse gas, is produced in rivers through a series of microbial metabolic pathways. However, the microbial source of N 2 O production and the degree of N 2 O reduction in river systems are not well understood and quantified. This work investigated isotopic compositions (δ 15 N-N 2 O and δ 18 O-N 2 O) and N 2 O site preference as well as N 2 O-related microbial features, thereby differentiating the importance of nitrification, denitrification, and N 2 O reduction in controlling N 2 O emissions from five rivers on the eastern Qinghai-Tibet Plateau (EQTP). The average N 2 O concentration in overlying water (15.2 nmol L −1 ) was close to that in porewater (17.5 nmol L −1 ), suggesting that both overlying water and sediment are potentially important sources of N 2 O. Canonical and nitrifier denitrification dominated riverine N 2 O production, with contribution being approximately 90%. Nitrification is a nonnegligible source of N 2 O production, and N 2 O concentration was positively correlated with nitrification genetic potential. The degree of N 2 O reduction ranged from 78.1 to 94.1% (averaging 90%), significantly exceeding the reported values (averaging 70%) in other freshwaters, which was attributed to the higher ratios of organic carbon to nitrogen and lower ratio of (nirS + nirK)/nosZ in EQTP rivers. This study indicates that a combination of isotopic and isotopocule values with functional microbe analysis is useful for quantifying the microbial sources of N 2 O in rivers, and the intense microbial reduction of N 2 O significantly accounts for the low N 2 O emissions observed in EQTP rivers, suggesting that both the production and consumption of N 2 O in rivers should be considered in the future.