The biomass of undisturbed tropical forests has likely increased in the past few decades, probably as a result of accelerated tree growth. Higher CO2 levels are expected to raise plant photosynthetic rates and enhance water-use efficiency, that is, the ratio of carbon assimilation through photosynthesis to water loss through transpiration. However, there is no evidence that these physiological responses do indeed stimulate tree growth in tropical forests. Here we present measurements of stable carbon isotopes and growth rings in the wood of 1,100 trees from Bolivia, Cameroon and Thailand. Measurements of carbon isotope fractions in the wood indicate that intrinsic water-use efficiency in both understorey and canopy trees increased by 30–35% over the past 150 years as atmospheric CO2 concentrations increased. However, we found no evidence for the suggested concurrent acceleration of individual tree growth when analysing the width of growth rings. We conclude that the widespread assumption of a CO2-induced stimulation of tropical tree growth may not be valid.