The intense human activities can cause irreversible environmental problems. Eucalyptus is a forest species widely used in planted forests, with a great capacity to assist in the mitigation of CO2 emissions and accumulation due to its C3 metabolism and high retention of carbon molecules in its biomass. The objective of this study was to investigate the differences in the sap proteome of two Eucalyptus species grown in an atmosphere enriched with CO2. For this purpose, young Eucalyptus grandis and Eucalyptus urophylla plants were grown in growth chambers 20 days under controlled atmospheric CO2 rates. The vascular proteome revealed 146 extracellular proteins, and their relative abundance was associated with the enriched atmosphere treatments. The analysis of protein function and abundance revealed that E. grandis proteins are mainly involved in organic substance metabolism and proteolysis, while less abundant proteins are related to cellular defense responses. Similar results were obtained for E. urophylla, with the most abundant proteins performing metabolic functions, while the least abundant protein was related to oxidative stress. These results may contribute to a better understanding of the mechanisms involved in the response of eucalyptus species to increased CO2 and provide useful information for the management and cultivation of these species in high levels of carbon dioxide environments.