How will forests respond to increases in atmospheric CO2 concentrations over the next century? This question is the subject of intensive research, and is based mostly on the results of greenhouse experiments, landscape-level experiments and modeling. In this paper, we examined leaf morphological and physiological characteristics of Nothofagus dombeyi populations exposed to different atmospheric CO2 levels by analyzing leaf cuticle from a Late Pleistocene (similar to 15,500-13,200 cal yr BP) fossil site, a Middle Holocene (similar to 6700-4900 cal yr BP) fossil site, and a modern (2016) collection in mid-latitude Chile. Atmospheric CO2 concentrations were inferred as similar to 230 ppm, similar to 270 ppm and similar to 400 ppm based on a comparison with icecore and instrumental records. Our findings show that as atmospheric CO2 concentrations increased by 74% during the transition from late glacial to modern conditions, stomatal density decreased by 24% and maximum stomatal conductance decreased by 42%. In contrast, we observed a 10% increase in stomatal size, a 195% increase in intrinsic water use efficiency, and an increase in leaf nitrogen content. Carbon discrimination decreased as CO2 levels increased contrary to our expectations underscoring that additional factors such as water availability and nutrients can affect carbon discrimination. We also tested the paleo-CO2 method of Franks that performed well under high CO2 conditions (similar to 400 ppm) but failed under low levels (similar to 230 ppm) of atmospheric CO2 indicating the need for a better understanding of photosynthesis during glacial times.