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The climate in the Amazon region is particularly sensitive to surface processes and properties such as heat fluxes and vegetation coverage. Rainfall is a key expression of the land surface–atmosphere interactions in the region due to its strong dependence on forest transpiration. While a large number of past studies have shown the impacts of large-scale deforestation on annual rainfall, studies on the isolated effects of elevated atmospheric CO2 concentrations (eCO2) on canopy transpiration and rainfall are scarcer. Here, for the first time, we systematically compare the plant physiological effects of eCO2 and deforestation on Amazon rainfall. We use the CPTEC Brazilian Atmospheric Model (BAM) with dynamic vegetation under a 1.5×CO2 experiment and a 100 % substitution of the forest by pasture grasslands, with all other conditions held similar between the two scenarios. We find that both scenarios result in equivalent average annual rainfall reductions (Physiology: −257 mm, −12 %; Deforestation: −183 mm, −9 %) that are above the observed Amazon rainfall interannual variability of 5 %. The rainfall decreases predicted in the two scenarios are linked to a reduction of approximately 20 % in canopy transpiration but for different reasons: the eCO2-driven reduction of stomatal conductance drives the change in the Physiology experiment, and the smaller leaf area index of pasturelands (−72 % compared to tropical forest) causes the result in the Deforestation experiment. The Walker circulation is modified in the two scenarios: in Physiology due to a humidity-enriched free troposphere with decreased deep convection due to the heightening of a drier and warmer (+2.1 ∘C) boundary layer, and in Deforestation due to enhanced convection over the Andes and a subsidence branch over the eastern Amazon without considerable changes in temperature (−0.2 ∘C in 2 m air temperature and +0.4 ∘C in surface temperature). But again, these changes occur through different mechanisms: strengthened west winds from the Pacific and reduced easterlies entering the basin affect the Physiology experiment, and strongly increased easterlies influence the result of the Deforestation experiment. Although our results for the Deforestation scenario agree with the results of previous observational and modelling studies, the lack of direct field-based ecosystem-level experimental evidence regarding the effect of eCO2 on moisture fluxes in tropical forests confers a considerable level of uncertainty to any projections of the physiological effect of eCO2 on Amazon rainfall. Furthermore, our results highlight the responsibilities of both Amazonian and non-Amazonian countries to mitigate potential future climatic change and its impacts in the region, driven either by local deforestation or global CO2 emissions.