The permanently stratified Lake Kivu is one of the largest freshwater reservoirs of dissolved methane (CH4) on Earth. Yet CH4 emissions from its surface to the atmosphere have been estimated to be 2 orders of magnitude lower than the CH4 upward flux to the mixed layer, suggesting that microbial CH4 oxidation is an important process within the water column. A combination of natural abundance stable carbon isotope analysis (δ13C) of several carbon pools and 13CH4-labelling experiments was carried out during the rainy and dry season to quantify (i) the contribution of CH4-derived carbon to the biomass, (ii) methanotrophic bacterial production (MBP), and (iii) methanotrophic bacterial growth efficiency (MBGE), defined as the ratio between MBP and gross CH4 oxidation. We also investigated the distribution and the δ13C of specific phospholipid fatty acids (PLFAs), used as biomarkers for aerobic methanotrophs. Maximal MBP rates were measured in the oxycline, suggesting that CH4 oxidation was mainly driven by oxic processes. Moreover, our data revealed that methanotrophic organisms in the water column oxidized most of the upward flux of CH4, and that a significant amount of CH4-derived carbon was incorporated into the microbial biomass in the oxycline. The MBGE was variable (2-50%) and negatively related to CH4 : O2 molar ratios. Thus, a comparatively smaller fraction of CH4-derived carbon was incorporated into the cellular biomass in deeper waters, at the bottom of the oxycline where oxygen was scarce. The aerobic methanotrophic community was clearly dominated by type I methanotrophs and no evidence was found for an active involvement of type II methanotrophs in CH4 oxidation in Lake Kivu, based on fatty acids analyses. Vertically integrated over the water column, the MBP was equivalent to 16-60% of the average phytoplankton particulate primary production. This relatively high magnitude of MBP, and the substantial contribution of CH4-derived carbon to the overall biomass in the oxycline, suggest that methanotrophic bacteria could potentially sustain a significant fraction of the pelagic food web in the deep, meromictic Lake Kivu.