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Abstract Kaolin deposits in the Seto-Tono district, central Japan, were formed by intense kaolinization of lacustrine arkose sediments deposited in small and shallow inland lakes in the late Miocene. Based on mineralogical and stable isotopic (Fe, C, N) studies of Motoyama kaolin deposit in the Seto area, we concluded that it was formed by microbial nitrification and acidification of lacustrine sediments underneath an inland lake. Small amounts of Fe–Ti oxides and Fe-hydroxide in the kaolin clay indicated that iron was oxidized and leached during the kaolinization. The field occurrences indicate that leached ferric iron precipitated on the bottom of the kaolin deposit as limonite crusts, and their significantly fractionated Fe isotope compositions suggest the involvement of microbial activity. The C/N ratios of most of the kaolin clay are distinctly higher than those of modern lacustrine sediment. Although, the possibility of a low-temperature hydrothermal origin of the kaolin deposit cannot be completely ruled out, it is more likely that acidification by dilute nitric acid formed from plant-derived ammonia could have caused the kaolinization, Fe oxidation and leaching. The nitrate-dependent microbial Fe oxidation is consistent with dilute nitric acid being the predominant oxidant.