It is commonly assumed that methane (CH4) released by lakes into the atmosphere is mainly produced in anoxic sediment and transported by diffusion or ebullition through the water column to the surface of the lake. In contrast to that prevailing idea, it has been gradually established that the epilimnetic CH4 does not originate exclusively from sediments but is also locally produced or laterally transported from the littoral zone. Therefore, CH4 cycling in the epilimnion and the hypolimnion might not be as closely linked as previously thought. We utilized a high-resolution method used to determine dissolved CH4 concentration to analyze a Siberian lake in which epilimnetic and hypolimnetic CH4 cycles were fully segregated by a section of the water column where CH4 was not detected. This layer, with no detected CH4, was well below the oxycline and the photic zone and thus assumed to be anaerobic. However, on the basis of a diffusion-reaction model, molecular biology, and stable isotope analyses, we determined that this layer takes up all the CH4 produced in the sediments and the deepest section of the hypolimnion. We concluded that there was no CH4 exchange between the hypolimnion (dominated by methanotrophy and methanogenesis) and the epilimnion (dominated by methane lateral transport and/or oxic production), resulting in a vertically segregated lake internal CH4 cycle.