We studied the oxidation and efflux of methane (CH₄) in a small, polyhumic lake, Mekkojärvi (southern Finland), during 6 weeks in autumn when the stability of the water mass first weakened, temporarily restabilized, and finally mixed completely. During the summer stratification period, CH₄ had accumulated in the anoxic hypolimnion to high concentrations (>150 mmol m⁻³). Gradual mixing of the water column during the autumn allowed access to both oxygen and CH₄ by aerobic methane-oxidizing bacteria (MOB) deeper in the water column. Thus the bulk (∼83–88%) of the CH₄ accumulated in the hypolimnion was subsequently consumed by MOB while only 12–17% was lost from the lake to the atmosphere at a rate varying between 0.05 and 8.8 mmol m⁻² d⁻¹. Phospholipid fatty acid (PLFA) analyses revealed that type I methanotrophs (MOB I) were responsible for this CH₄ oxidation. The stable carbon isotope ratio of CH₄ in the water column (δ ¹³C-CH₄) ranged from −81.2‰ close to the bottom to −45.1‰ at the surface. At CH₄ concentrations >1 mmol m⁻³ the δ ¹³C-CH₄ isotopic value was linearly related to the specific oxidation rate. The carbon isotopic fractionation factor α for aerobic CH₄ oxidation calculated from the results (1.037) is within the range of maximum values reported in literature. Our results show that in stratified boreal lakes the greatest MOB activity and effluxes of CH₄ both occur during autumnal mixing. Owing to their great abundance and areal cover, small water bodies should not be neglected in global analyses of the CH₄ cycle.