A 450 cm sediment core spanning the last 3,500 years was collected from 4.6 m of water in Lake Infernão, located in the floodplain of the Moji-Guaçu River in SE Brazil, to reconstruct the wetland system (river and lake) paleohydrology. A multidisciplinary approach using isotopic (¹³C and ¹⁵N), chemical (C/N ratio and lignin derived phenols) and physical analyses (% water content and density) were used in this study. A coarse sand unit in the lower part of the core (450–417 cm) corresponds to the paleoriver. The rest of the sediment core (dark clay) shows a gradual change from a fluvial to a lacustrine environment. ¹⁴C dates from the lower part of the core (417–200 cm), demonstrate an inversion and narrow span in age (3,000–3,500 yrs B.P.) corresponding to a period of rapid fluvial sedimentation and reworking. The lake seems to have been well established only after 3,000 yrs B.P. The change from a fluvial to a lacustrine environment is inferred from the isotopic and chemical data. Sediment of the fluvial phase is characterized by higher ¹³C values, lower C and N content and a lignin content representative of woody-tissues. Sediment of the lacustrine phase show a gradual decrease in ¹³C values toward the top of the core reflecting the influence of organic carbon derived by phytoplankton and aquatic macrophytes (C₃ plants). The trend to higher C and N content and low C/N ratios also support this interpretation. The lower lignin content indicates increase contributions of non-vascular plants (phytoplankton). Similarly, the slight increase of non-woody tissues (higher C/V ratios) and the predominance of angiosperm plants (high S/V ratios) as organic matter source indicate an increase contribution of aquatic plants during the lacustrine phase. Our data presented here in addition to the geomorphological information and the available paleoclimatic scenario of pollen-inferred analyses for aquatic systems from the southeastern Brazil point to a climatic origin for this fluvial-lacustrine transition at about 3,000 yrs B.P. and to a climatic evolution leading to the present conditions and to a consequently gradual isolation of Lake Infernão from the influence of the Moji-Guaçu River.