Tonle Sap Lake, Cambodia, possesses one of the most productive inland fisheries in the world and is a vital natural resource for the country. The lake is connected to the Mekong River via the Tonle Sap River. Flow in the Tonle Sap River reverses seasonally, with water exiting the lake in the dry season and entering the lake during the summer monsoon. This flood pulse drives the lake’s biological productivity. We used Sr, Nd, and Pb isotopes and elemental concentrations in lake sediment cores to track changes in the provenance of deposits in Tonle Sap Lake. We sought to determine when the lake first began to receive water and sediment input via the Mekong River, which initiated flood pulse processes. The transition from a non-pulsing lake to the Mekong-connected system is marked by shifts to values of 87Sr/86Sr, εNd, and 207Pb/204Pb that are characteristic of Mekong River sediments. In addition, magnetic susceptibility increased and sediment elemental composition changed. Elemental (P) measures point to enhanced phosphorus loading and C/N and isotope ratios of bulk organic matter indicate a shift to greater relative contribution of organic material from aquatic versus terrestrial environments, coinciding with the initiation of flood pulse processes. On the basis of radiocarbon dating in two cores, we estimate the initiation of the annual flood pulse occurred between 4,450 and 3,910 cal year BP