An expressed sequence tag (EST) highly similar to BdPT1-4, a phosphate transporter (PT) gene in Brachypodium distachyon, was obtained in a wheat root cDNA subtractive suppression library containing genes that respond to low-phosphate (Pi) stress. The DNA sequence covering this EST (designated as TaPht1;4) was determined based on screening a wheat DNA library. TaPht1;4 consists of two exons and one intron and encodes a 555 amino acid (aa) polypeptide with a molecular weight of 60.85kDa and an isoelectric point of 7.60. TaPht1;4 contains 12 conserved membrane-spanning domains similar to previously reported PTs in diverse plant species. Yeast complement analysis in low-Pi medium confirmed that TaPht1;4 confers the capacity to uptake Pi to MB192, a yeast strain with a defective high-affinity PT; with an apparent Km of 35.3μM. The TaPht1;4 transcripts were specifically detected in the root and were highly induced under Pi deficiency. TaPht1;4 was also expressed following a diurnal pattern, i.e. high levels during daytime and low levels during night-time. TaPht1;4 overexpression and downregulation dramatically altered the plant phenotypic features under low-Pi conditions. Samples that overexpressed TaPht1;4 had significantly improved growth traits and accumulated more Pi than the wild-type plant and those with downregulated TaPht1;4 expression. Therefore, TaPht1;4 is a high-affinity PT gene that plays a critical role in wheat Pi acquisition under Pi deprivation.