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Barley (Hordeum vulgare) is more drought tolerant than other cereals, thus making it an excellent model for the study of the chemical, transcriptomic and physiological effects of water deficit. Roots are the first organ to sense soil water deficit. Therefore, we studied the response of barley seminal roots to different water potentials induced by polyethylene glycol (PEG) 8000. We investigated changes in anatomical parameters by histochemistry and microscopy, quantitative and qualitative changes in suberin composition by analytical chemistry, transcript changes by RNA-sequencing (RNA-Seq), and the radial water and solute movement of roots using a root pressure probe. In response to osmotic stress, genes in the suberin biosynthesis pathway were upregulated that correlated with increased suberin amounts in the endodermis and an overall reduction in hydraulic conductivity (Lpr). In parallel, transcriptomic data indicated no or only weak effects of osmotic stress on aquaporin expression. These results indicate that osmotic stress enhances cell wall suberization and markedly reduces Lpr of the apoplastic pathway, whereas Lpr of the cell-to-cell pathway is not altered. Thus, the sealed apoplast markedly reduces the uncontrolled backflow of water from the root to the medium, whilst keeping constant water flow through the highly regulated cell-to-cell path.