Water and its influence on plants likely exerted strong adaptive pressures in human evolution. Understanding relationships among water, plants, and early humans is limited both by incomplete terrestrial records of environmental change and by indirect proxy data for water availability. Here we present a continuous record of stable hydrogen-isotope compositions (expressed as δD values) for lipid biomarkers preserved in lake sediments from an Early Pleistocene archaeological site in eastern Africa—Olduvai Gorge. We convert sedimentary leaf- and algal-lipid δD values into estimates for ancient source-water δD values by accounting for biochemical, physiological, and environmental influences on isotopic fractionation via published water–lipid enrichment factors for living plants, algae, and recent sediments. Reconstructed precipitation and lakewater δD values, respectively, are consistent with modern isotopic hydrology and reveal that dramatic fluctuations in water availability accompanied ecosystem changes. Drier conditions, indicated by less negative δD values, occur in association with stable carbon-isotopic evidence for open, C4-dominated grassland ecosystems. Wetter conditions, indicated by lower δD values, are associated with expanded woody cover across the ancient landscape. Estimates for ancient precipitation amounts, based on reconstructed precipitation δD values, range between approximately 250 and 700 mm·y−1 and are consistent with modern precipitation data for eastern Africa. We conclude that freshwater availability exerted a substantial influence on eastern African ecosystems and, by extension, was central to early human proliferation during periods of rapid climate change.