In eastern North America, large prehistoric settlements were concentrated in and along the floodplains of the midcontinent, but few sedimentary records have been examined adjacent to these sites to evaluate the impacts of Native American land use on terrestrial ecosystems. Here we report a high-resolution and multiproxy paleoecological record from Horseshoe Lake, an oxbow lake in the central Mississippi River valley that is adjacent to the Cahokia site (Illinois, USA), the largest prehistoric settlement north of Mexico. Palynological and carbon isotope data document pronounced vegetation changes over the past 1700 yr driven primarily by land use, including 900 yr (450–1350 CE) of sustained prehistoric human impacts. Rapid forest clearance was followed closely by the proliferation of indigenous seed crops of the Eastern Agricultural Complex beginning ca. 450 CE, centuries before the emergence of Cahokia at 1050 CE. Agricultural intensification that included the use of maize (Zea mays subsp. mays) followed this initial clearance, with peak land use intensity between 900 and 1200 CE. A large flood event ca. 1200 CE marks the onset of agricultural contraction and Cahokia’s decline. Reforestation follows the abandonment of the Cahokia region at ca. 1350 CE. The Horseshoe Lake record thus indicates that regional agricultural activity began abruptly at 450 CE and intensified over the following centuries, well before the formation of Cahokia and other large prehistoric settlements. The evidence that a major flood coincided with the onset of Cahokia’s decline is noteworthy, but will require corroboration from additional records.