Soil moisture heterogeneity in the root-zone is common during both the establishment of tree seedlings and in experiments aiming to impose semi-constant soil moisture deficits, but its effects on regulating plant water use compared to homogenous soil drying are not well known in trees. Pronounced vertical soil moisture heterogeneity was imposed on black poplar (Populus nigra L.) grown in soil columns by altering irrigation frequency, to test whether plant water use, hydraulic responses, root phytohormone concentrations, and root xylem sap chemical composition differed between wet (well-watered, WW), homogeneously (infrequent deficit irrigation, IDI) and heterogeneously dry soil (frequent deficit irrigation, FDI). At the same bulk soil water content, FDI plants had greater water use than IDI plants, probably because root abscisic acid (ABA) concentration was low in the upper wetter layer of FDI plants, which maintained root xylem sap ABA concentration at basal levels in contrast with IDI. Soil drying did not increase root xylem concentration of any other hormone. Nevertheless, plant-to-plant variation in xylem jasmonic acid (JA) concentration was negatively related to leaf stomatal conductance within WW and FDI plants. However, feeding detached leaves with high (1,200 nM) JA concentrations via the transpiration stream decreased transpiration only marginally. Xylem pH and sulphate concentration decreased in FDI plants compared to well-watered plants. Frequent deficit irrigation increased root accumulation of the cytokinin trans-zeatin (tZ), especially in the dry lower layer, and of the ethylene precursor ACC, in the wet upper soil layer. Root hormone accumulation might explain the maintenance of high root hydraulic conductance and water use in FDI plants (similar to well-watered plants) compared to IDI plants. In irrigated tree crops, growers could vary irrigation scheduling to control water use by altering the hormone balance.