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Human impacts prior to the Industrial Revolution are not well constrained. We investigate whether the decline in global atmospheric CO2 concentration by 7-10 ppm in the late 1500s and early 1600s which globally lowered surface air temperatures by 0.15 degrees C, were generated by natural forcing or were a result of the large-scale depopulation of the Americas after European arrival, subsequent land use change and secondary succession. We quantitatively review the evidence for (i) the pre-Columbian population size, (ii) their per capita land use, (iii) the post-1492 population loss, (iv) the resulting carbon uptake of the abandoned anthropogenic landscapes, and then compare these to potential natural drivers of global carbon declines of 7-10 ppm. From 119 published regional population estimates we calculate a pre-1492 CE population of 60.5 million (interquartile range, IQR 44.8-78.2 million), utilizing 1.04 ha land per capita (IQR 0.98-1.11). European epidemics removed 90% (IQR 87-92%) of the indigenous population over the next century. This resulted in secondary succession of 55.8 Mha (IQR 39.0-78.4 Mha) of abandoned land, sequestering 7.4 Pg C (IQR 4.9-10.8 Pg C), equivalent to a decline in atmospheric CO2 of 3.5 ppm (IQR 2.3-5.1 ppm CO2). Accounting for carbon cycle feedbacks plus LUC outside the Americas gives a total 5 ppm CO2 additional uptake into the land surface in the 1500s compared to the 1400s, 47 -67% of the atmospheric CO2 decline. Furthermore, we show that the global carbon budget of the 1500s cannot be balanced until large-scale vegetation regeneration in the Americas is included. The Great Dying of the Indigenous Peoples of the Americas resulted in a human-driven global impact on the Earth System in the two centuries prior to the Industrial Revolution. (C) 2019 The Authors. Published by Elsevier Ltd.