Processes linked to woody plant encroachment in grassland are still not well understood, especially the interactions between trees and soil water availability. Our aim was to study the depth of water uptake by trees in grassland and its controlling factors. We studied water uptake in Vachellia sieberiana trees (hereafter called 'acacias') in a humid grassland of South Africa. We especially studied the effect of size classes of acacias and their position in the catena on water potential (ψ) of acacia leaves and water uptake depth of acacias across seasons, as inferred from isotope δ 18 O values of soil and stem water samples. Acacias were more water stressed in February (wet season) with the most negative water potentials. Taller acacias (>3 m height) were more water-stressed (ψ ± SD = −1.62 ± 0.81 MPA) than smaller acacias (<1 m height) (ψ ± SD = −1.32 ± 0.80 MPA). In September (end of dry season), all tree size classes uptake water equally in A and B soil horizons. In February (middle of the wet season), tall, medium and even small trees favoured the B horizon. Small trees had similar plasticity in water uptake to taller trees and could modify depth of water uptake between seasons. This process could increase their probability of becoming adults, and may favour tree encroachment in grasslands.