Abstract Plants growing in dense canopies are exposed to vertical light gradients and show photosynthetic acclimation at the whole-plant level, resulting in efficient photosynthetic carbon gain. We studied the role of cytokinins transported through the transpiration stream as one of probably multiple signals for photosynthetic acclimation to light gradients using both tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). We show that substantial variation in leaf transpiration parallels the light gradient in tobacco canopies and experimental reduction of the transpiration rate of a leaf, independent of light, is sufficient to reduce photosynthetic capacity in both species, as well as transcript levels of the small subunit of Rubisco (rbcS) gene in Arabidopsis. Mass spectrometric analysis of xylem sap collected from intact, transpiring tobacco plants revealed that shaded leaves import less cytokinin than leaves exposed to high light. In Arabidopsis, reduced transpiration rate of a leaf in the light is associated with lower cytokinin concentrations, including the bioactive trans-zeatin and trans-zeatin riboside, as well as reduced expression of the cytokinin-responsive genes ARR7 and ARR16. External application of cytokinin to shaded leaves rescued multiple shade effects, including rbcS transcript levels in both species, as did locally induced cytokinin overproduction in transgenic tobacco plants. From these data, we conclude that light gradients over the foliage of a plant result in reduced cytokinin activity in shaded leaves as a consequence of reduced import through the xylem and that cytokinin is involved in the regulation of whole-plant photosynthetic acclimation to light gradients in canopies.