Megafauna paintings have accompanied the earliest archaeological contexts across the continents, revealing a fundamental inter-relationship between early humans and megafauna during the global human expansion as unfamiliar landscapes were humanized and identities built into new territories. However, the identification of extinct megafauna from rock art is controversial. Here, we examine potential megafauna depictions in the rock art of Serranía de la Lindosa, Colombian Amazon, that includes a giant sloth, a gomphothere, a camelid, horses and three-toed ungulates with trunks. We argue that they are Ice Age rock art based on the (i) naturalistic appearance and diagnostic morphological features of the animal images, (ii) late Pleistocene archaeological dates from La Lindosa confirming the contemporaneity of humans and megafauna, (iii) recovery of ochre pigments in late Pleistocene archaeological strata, (iv) the presence of most megafauna identified in the region during the late Pleistocene as attested by archaeological and palaeontological records, and (v) widespread depiction of extinct megafauna in rock art across the Americas. Our findings contribute to the emerging picture of considerable geographical and stylistic variation of geometric and figurative rock art from early human occupations across South America. Lastly, we discuss the implications of our findings for understanding the early human history of tropical South America. This article is part of the theme issue 'Tropical forests in the deep human past'.