The discovery of methane (CH4) accumulation in oxic marine and limnic waters has redefined the role of aquatic environments in the regional CH4 cycle. Although CH4 accumulation in oxic surface waters became apparent in recent years, the sources are still subject to controversial discussions. We present high-resolution in situ measurements of CH4 concentration and its stable isotope composition in a stratified mesotrophic lake. We show that CH4 accumulation in surface waters originates from a highly dynamic interplay between (oxic) CH4 production and emission to the atmosphere. Laboratory incubations of different phytoplankton types and application of stable isotope techniques provide a first unambiguous evidence that major phytoplankton classes in Lake Stechlin per se produce CH4 under oxic conditions. Combined field and lab results show that the photoautotroph community is an important driver for CH4 production and its highly dynamic accumulation in oxic surface waters.