The North American prairie-forest border is a major biogeographic boundary ultimately determined by the macroclimate. Climate variability during the Holocene affected the vegetation in this area, but impacts on human paleodemography are unknown. At a regional scale, community structure is partly determined by fire, however the extent to which anthropogenic burning has affected fire regimes over the Holocene is unresolved. This study investigates the interaction between climate variability, vegetation changes, fire regimes, and human population levels in the North American prairie-forest ecotone during the Holocene using information from publically-available paleoenvironmental databases. Biomass burning was associated with moisture and vegetation more than with human population size, suggesting anthropogenic burning did not significantly influence the composition and location of the prairie-forest border. Human population growth rates were impacted by sociocultural developments and environmental changes, with most changes in subsistence strategies occurring during climate regime shifts. The development of the Eastern Agricultural Complex (5.0 – 3.8 ka) and the transition to more mesic conditions after 4.0 ka facilitated long-term population growth. The arrival of maize and the bow-and-arrow at 2.2 and 1.6 ka, respectively, resulted in increased population growth, and after 1 ka, maize agriculture intensification, aided by a warmer climate, accelerated population growth. The collapse of the city of Cahokia is linked to a wider population decline across the Midwest precipitated by the Medieval Warm Period – Little Ice Age transition. Populations across a significant portion of North America were in decline at the time of European colonization. These findings provide evidence against a large-scale early Anthropocene in North America, and illustrate the importance of climate change in influencing human history. •Holocene population levels were impacted by sociocultural and environmental changes.•No evidence of human regional-scale impact on prairie-forest border or fire regime.•Cahokia’s collapse occurred during population declines across the Midwest.•Population across much of North America was decreasing prior to European arrival.