Tropical East African Lake Tanganyika hosts the Earth's largest anoxic freshwater body. The entire water column holds over 23 Tg of the potent greenhouse gas methane (CH4). Methane is formed under sulphate-poor conditions via carbon dioxide reduction or fermentation from detritus and relict sediment organic matter. Permanent density stratification supports an accumulation of CH4 below the permanent oxycline. Despite CH4 significance for global climate, anaerobic microbial consumption of CH4 in freshwater is poorly understood. Here we provide evidence for intense methanotrophic activity not only in the oxic but also in the anoxic part of the water column of Lake Tanganyika. We measured CH4, 13C of dissolved CH4, dissolved oxygen (O2), sulphate (SO42−), sulphide (HS−) and the transient tracers chlorofluorocarbon-12 (CFC-12) and tritium (3H). A basic one-dimensional model, which considers vertical transport and biogeochemical fluxes and transformations, was used to interpret the vertical distribution of these substances. The results suggest that the anaerobic oxidation of CH4 is an important mechanism limiting CH4 to the anoxic zone of Lake Tanganyika. The important role of the anaerobic oxidation for CH4 concentrations is further supported by high abundances (up to ∼33% of total DAPI-stained cells) of single living archaea, identified by fluorescence in situ hybridization.