This paper reports manganese (Mn) fractionation in samples collected from the water column and sediments in an environmental protection area in the Alto do Paranapanema Basin (São Paulo State, Brazil). The three locations studied showed equivalent Mn levels, with moderate seasonal differences (p < 0.05). The sediment samples contained five Mn species (p < 0.05): iron and manganese (hydr)oxides > Mn bound to carbonates ≈ exchangeable Mn ≈ Mn bound to silicates > Mn bound to organic matter (p < 0.05). The water samples contained three species (p < 0.05): particulate Mn > labile Mn ≈ non-labile Mn. The data suggest that Mn has a natural origin (Enrichment Factor EF < 2; Geoaccumulation Index Igeo < 0) and moderate environmental risk (Risk Assessment Code RAC ∼ 30%). At the same time, under certain conditions some manganese species could be present in a state of equilibrium between the water column and sediment. These results could provide a basis for Mn management in the Alto do Paranapanema Basin.