Low concentrations of boron (B) in the external medium can induce uptake mechanisms whereby plants can develop a concentration gradient for B against the external medium. These mechanisms seem to be widespread among herbaceous species. In this study, olive (Olea europaea L.) plants were acclimated to either high (23 μm; controls) or low (0.5 μm; low-B plants) concentrations of B for 45 d, in a hydroponic culture. Afterwards, a 7-h uptake experiment was conducted by transferring plants of both groups to a series of nutrient solutions with B concentrations ranging from 0.5 to 23 μm. Analysis of B concentration in cell sap of root and xylem exudate was performed by the borate–chromotropic acid HPLC assay. Plants acclimated to high-B concentration showed root cell and xylem exudate B concentrations that were comparable to those of the external medium. In contrast, plants acclimated to low-B concentration were able to develop concentrations of B in root cells up to 2-fold higher than those of the external medium. Moreover, B concentrations in xylem exudate for both plant groups corresponded to those of the root cell sap, indicating diffusion equilibrium. These results support the existence of a mechanism that concentrates B in the root cell sap against the nutrient solution when olive plants are acclimated to low-B conditions.