The flux of methane (CH4) from inland waters to the atmosphere has a profound impact on global atmospheric green- house gas (GHG) levels, and yet, strikingly little is known about the dynamics controlling sources and sinks of CH4 in the aquatic setting. Here, we examine the cycling and flux of CH4 in six large rivers in the Amazon basin, including the Amazon River. Based on stable isotopic mass balances of CH4, inputs and outputs to the water column were esti- mated. We determined that ecosystem methane oxidation (MOX) reduced the diffusive flux of CH4 by approximately 28–96% and varied depending on hydrologic regime and general geochemical characteristics of tributaries of the Amazon River. For example, the relative amount of MOX was maximal during high water in black and white water rivers and minimal in clear water rivers during low water. The abundance of genetic markers for methane-oxidizing bacteria (pmoA) was positively correlated with enhanced signals of oxidation, providing independent support for the detected MOX patterns. The results indicate that MOX in large Amazonian rivers can consume from 0.45 to 2.07 Tg CH4/yr, representing up to 7% of the estimated global soil sink. Nevertheless, climate change and changes in hydrology, for example, due to construction of dams, can alter this balance, influencing CH4 emissions to atmosphere.