AbstractDeficiency of phosphate (Pi) is one of the main growth-limiting factors for crops. Generally, phosphate transporters play a key role in the uptake of P in the crops. However, current knowledge regarding the molecular mechanism underlying Pi transport is still limited. In this study, a phosphate transporter (PT) gene, designatedHvPT6, was isolated from a cDNA library constructed from hulless barley “Kunlun 14.” The promoter of HvPT6 showed a large number of elements related to plant hormones. The expression pattern also indicated thatHvPT6 was highly induced by low phosphorus, drought, abscisic acid, methyl jasmonate and gibberellin. Phylogenetic tree analysis revealed that HvPT6 belongs to the same subfamily of the major facilitator superfamily as OsPT6 fromOryza sativa. Subcellular localization of HvPT6:GFP using transient expression ofAgrobacterium tumefaciensshowed the green fluorescent protein signal in the membrane and nucleus of theNicotiana benthamianaleaves. Overexpressing HvPT6 led to a longer and higher lateral root length and dry matter yield in the transgenicArabidopsislines under low Pi conditions, indicating that HvPT6 improves plant tolerance under Pi-deficient conditions. This study will lay a molecular basis for phosphate absorption mechanism in barley and breeding barley with high-efficient phosphate uptake.