Since the Aha Lake, located in Guizhou Province, China, was enlarged in 1982, a great amount of iron and manganese were input into it owing to the uninterrupted pollution from acidic mining drainage of coal mines (AMD). The results show that dissolved Fe2+ and Mn2+, mainly from AMD, were markedly separated each other as water flowed from inlet to downstream. The Aha lake water, with the average pH equal to 7.28, was not acidified just because the lake is developed in the karst area and people continuously put lime in it in recent years. Fe2 + and Mn2 + deposit in the different locations in lake because of the difference of oxidizing rate between Fe2+ and Mn2+ during water flowing from inlet to downstream, and also forming the horizontal separation along the lake water flowing direction. Manganese tends to be obviously enriched in the upper of sediment in lake's deep locations, presenting the remarkable separation with iron in the vertical section. After normalized by Ti content in sediment, it is obvious that the remarkable separation between iron and manganese corresponds with the enlargement of lake in 1982. As shown in the concentration profile of pore water in sediment, iron was remobilized weakly while manganese reduction mainly happens in the upper of sediment, indicating its strong remobilization. Based on the studies, a mechanism for environment and fractionation of Fe2+ /Mn2 + during water flowing in the lake was also demonstrated.