Hypolimnetic oxygenation is a technique used to restore oxic conditions in the summer hypolimnia of lakes, thereby reducing internal P recycling, metal flux, and increasing fish habitat. O2 at the sediment–water interface plays a critical role in regulation of CH4 flux from lakes. Sediment microorganisms responsible for both methanogenesis and oxidation uniquely discriminate against 13C. Therefore, stable isotope analysis (SIA) can be used to estimate CH4-derived biomass in aquatic food webs. For example, investigators have used SIA to show that methane-oxidizing bacteria (MOB) may have high dietary importance for Chironomus in anaerobic and hypoxic environments. We proposed that oxygenation would reduce CH4 production and subsequent synthesis of CH4-derived biomass in Chironomus larvae, decreasing the influence of CH4 pathways to overall aquatic food webs. We tested our hypothesis in mesotrophic lakes with and without oxygenation. Chironomus larvae were 13C-depleted compared to available organic matter in treated and untreated lakes, signifying that MOB consumption may be an important energy source for these organisms. However, our hypothesis was refuted. Larvae from lakes with oxygenation displayed higher dietary MOB contribution than larvae from the untreated lake. Our results suggest oxygenation may alter chemotrophic energy flow within aquatic and linked terrestrial food webs.