While modern forests have their origin in the diversification and expansion of angiosperms in the Late Cretaceous and early Cenozoic, it is unclear whether the rise of closed-canopy tropical rainforests preceded or followed the end-Cretaceous extinction. The “canopy effect” is a strong vertical gradient in the carbon isotope (δ13C) composition of leaves in modern closed-canopy forests that could serve as a proxy signature for canopy structure in ancient forests. To test this, we report measurements of the carbon isotope composition of nearly 200 fossil angiosperm leaves from two localities in the Paleocene Cerrejón Formation and one locality in the Maastrichtian Guaduas Formation of Colombia. Leaves from one Cerrejón fossil assemblage deposited in a small fluvial channel exhibited a 6.3‰ range in δ13C, consistent with a closed-canopy forest. Carbon isotope values from lacustrine sediments in the Cerrejón Formation had a range of 3.3‰, consistent with vegetation along a lake edge. An even-narrower range of δ13C values (2.7‰) was observed for a leaf assemblage recovered from the Cretaceous Guaduas Formation, and suggests vegetation with an open canopy structure. Carbon isotope fractionation by Late Cretaceous and early Paleogene leaves was in all cases similar to that by modern relatives, consistent with estimates of low atmospheric CO2 during this time period. This study confirms other lines of evidence suggesting that closed-canopy forests in tropical South America existed by the late Paleocene, and fails to find isotopic evidence for a closed-canopy forest in the Cretaceous.