Maintaining low intracellular Na+ concentrations is an essential physiological strategy in salt stress tolerance in most cereal crops. Here, we characterized a member of the high-affinity K+ transporter (HKT) family in barley (Hordeum vulgare), HvHKT1;5, which negatively regulates salt tolerance and has different functions from its homology in other cereal crops. HvHKT1;5 encodes a plasma membrane protein localized to root stele cells, particularly in xylem parenchyma cells adjacent to the xylem vessels. Its expression was highly induced by salt stress. Heterogenous expression of HvHKT1;5 in Xenopus laevis oocytes showed that HvHKT1;5 was permeable to Na+, but not to K+, although its Na+ transport activity was inhibited by external K+. HvHKT1;5 knockdown barley lines showed improved salt tolerance, a dramatic decrease in Na+ translocation from roots to shoots, and increases in K+/Na+ when compared with wild-type plants under salt stress. The negative regulation of HvHKT1;5 in salt tolerance distinguishes it from other HKT1;5 members, indicating that barley has a distinct Na+ transport system. These findings provide a deeper understanding of the functions of HKT family members and the regulation of HvHKT1;5 in improving salt tolerance of barley.