Iron is an abundant element in the environment and acts as a thermodynamically favorable electron acceptor driving the anaerobic oxidation of methane (AOM). Presently, the role and environmental regulation of iron-driven AOM in rivers, an important source of methane emission, are nearly unknown. Here, we provided direct evidence for iron-driven AOM activity in sediment of a mountainous river (Wuxijiang River, China) through 13 C-labeled isotopic experiment. The potential rate of iron-driven AOM ranged between 0.40 and 1.84 nmol 13 CO 2 g (sediment) d -1 , which contributed 36% on average to total AOM activity when combined the potential nitrate- and nitrite-driven AOM rates measured previously. There were significant variations in iron-driven AOM rates among different reaches (upper, middle, and lower) and between seasons (summer and winter). Sediment temperature, pH, and nitrate content were closely associated with the dynamic of AOM activity. Our results indicate that iron-driven AOM has great potential for reducing methane emissions from riverine ecosystems, and suggest the necessity of taking both spatial and temporal scales into account to evaluate the quantitative role of this AOM process.