The emergence of forests on Earth (~385 million years ago, Ma)1 has been linked to an order-of-magnitude decline in atmospheric CO2 levels and global climatic cooling by altering continental weathering processes, but observational constraints on atmospheric CO2 before the rise of forests carry large, often unbound, uncertainties. Here, we calibrate a mechanistic model for gas exchange in modern lycophytes and constrain atmospheric CO2 levels 410–380 Ma from related fossilized plants with bound uncertainties of approximately ±100 ppm (1 sd). We find that the atmosphere contained ~525–715 ppm CO2 before continents were afforested, and that Earth was partially glaciated according to a palaeoclimate model. A process-driven biogeochemical model (COPSE) shows the appearance of trees with deep roots did not dramatically enhance atmospheric CO2 removal. Rather, shallow-rooted vascular ecosystems could have simultaneously caused abrupt atmospheric oxygenation and climatic cooling long before the rise of forests, although earlier CO2 levels are still unknown
