© 2019 Elsevier B.V. Methanogenesis in freshwater lakes has classically been considered to arise from anaerobic methanogens in oxygen-depleted sediments. However, the accumulation of supersaturated methane in fully oxygenated water columns is commonly observed in many lakes, and factors responsible for the formation of the subsurface methane maximum (SMM) remain largely unknown. The present study conducted in 14 Japanese freshwater lakes showed that the SMM formation during the summer stratification period is a common feature in large and deep oligotrophic lakes. The seasonal survey of a deep oligotrophic lake revealed that SMM formation may be uncoupled with the dissolution of atmospheric methane, as well as with the transport of methane from tributary rivers, littoral sediments, and hypolimnetic anoxic sources, suggesting the contribution of in situ methane production. In fact, batch-culture experiments confirmed that bacterioplankton present in lake subsurface waters produce methane aerobically through the decomposition of methylphosphonic acid. Moreover, the development of SMM was closely associated with the seasonal dynamics of planktonic cyanobacteria such as Synechococcus, which may carry the enzyme to catabolize organophosphonate compounds. Therefore, we suggest that the predominance of Synechococcus during the thermal stratification period plays a significant role in SMM formation, and likely the methane flux from lakes to the atmosphere.