Recent geological studies demonstrate that the Isthmus of Panama emerged some 10 m.y. earlier than previously assumed. Although absent today in Panama, Central American savanna environments likely developed in connection with the onset of Northern Hemisphere glaciations. As is widely recognized, most of the mammals crossing the isthmus since 2.5 Ma lived in savannas. Could climate-induced vegetational changes across Panama explain the delayed migration of mammals, rather than terrestrial connectivity? We investigate the congruence between cross-continental mammal migration and climate change through analysis of fossil data and molecular phylogenies. Evidence from fossil findings shows that the vast majority of mammals crossed between South and North America after ca. 3 Ma. By contrast, dated mammal phylogenies suggest that migration events started somewhat earlier, ca. 4–3 Ma, but allowing for biases toward greater ages of molecular than geologic dating and uncertainties in the former, we consider this age range not to be significantly earlier than 3 Ma. We conclude that savanna-like environments developed in response to the vast Laurentide ice sheet at the first Quaternary glaciation triggered the initiation of the Great American Biotic Interchange in mammals.