Recent investigations have shown that biogenic methane can be a carbon source for macro invertebrates in freshwater food webs. Stable carbon isotopic signatures, used to infer an organism's food source, indicated that methane can play a major role in the nutrition of chironomid larvae. However, the pathway of methane-derived carbon into invertebrate biomass is still not confirmed. It has been proposed that chironomid larvae ingest methane-oxidizing bacteria (MOB), but this has not been experimentally demonstrated to date. Using 13C-labelled methane we could show for the first time that chironomid larvae assimilate methane-derived carbon through MOB. Chironomid larval biomass was significantly 13C-enriched after dwelling for 10 days in lake sediment enriched with labelled methane. Moreover, phospholipid fatty acids diagnostic for MOB were detected in larval tissue and were significantly 13C-enriched, which encompasses the 13C-uptake predicted for a methane-based nutrition. Additionally, chironomid larvae fed on sediment and water-column derived MOB biomass.