The origin of methane discharged at ancient methane seeps is a key to understand subsurface fluid-flow processes in sedimentary basins. The carbon isotopic composition of methane-seep carbonates can be used to estimate the origin of methane, but mixing of multiple carbon sources results in ambiguous interpretation. We here constrained the origin of methane at ancient seeps by using the carbon isotopic composition and the isotope fractionation of lipid biomarkers of anaerobic methane-oxidizing archaea. We estimated the carbon-isotope fractionation between methane and biomarkers as the offset between their delta C-13 values at modern seep sites in the Japan Sea and other regions of the world. The biomarker pentamethylicosane (PMI) extracted from a modern seep carbonate of the Japan Sea shows a 44 parts per thousand offset from the seep methane. On the basis of literature data, we calculated the carbon-isotope offset as mostly similar to 20-60 parts per thousand for PMI at modern seeps across the world. We also assessed the relationship of the delta C-13 values between methane and biomarkers by regression analysis on the data taken at the modern seeps, obtaining regression formulas to estimate the delta C-13 values of methane from those of biomarkers. For ancient seeps, we examined Miocene to Pleistocene methane-seep carbonate rocks collected from Japan Sea sediments. PMI extracted from these ancient carbonates have delta C-13 values ranging from -137 parts per thousand to -93 parts per thousand. Using the delta C-13 values of PMI and the carbon-isotope offset and regression formulas, we estimated that the methane at the ancient seeps was mainly of biogenic origin and migrated from the shallow subsurface.