Several phosphate transporters (PTs) that belong to the Pht2 family have been released in bioinformatics databases, but only a few members of this family have been functionally characterized. In this study, we found that wheat TaPHT2;1 shared high identity with a subset of Pht2 in diverse plants. Expression analysis revealed that TaPHT2;1 was strongly expressed in the leaves, was up-regulated by low Pi stress, and exhibited a circadian rhythmic expression pattern. TaPHT2;1-green fluorescent protein fusions in the leaves of tobacco and wheat were specifically detected in the chloroplast envelop. TaPHT2;1 complemented the Pi transporter activities in a yeast mutant with a defect in Pi uptake. Knockdown expression of TaPHT2;1 significantly reduced Pi concentration in the chloroplast under sufficient (2 mM Pi) and deficient Pi (100 μM Pi) conditions, suggesting that TaPHT2;1 is crucial in the mediation of Pi translocation from the cytosol to the chloroplast. The down-regulated expression of TaPHT2;1 resulted in reduced photosynthetic capacities, total P contents, and accumulated P amounts in plants under sufficient and deficient Pi conditions, eventually leading to worse plant growth phenotypes. The TaPHT2;1 knockdown plants exhibited pronounced decrease in accumulated phosphorus in sufficient and deficient Pi conditions, suggesting that TaPHT2;1 is an important factor to associate with a distinct P signaling that up-regulates other PT members to control Pi acquisition and translocation within plants. Therefore, TaPHT2;1 is a key member of the Pht2 family involved in Pi translocation, and that it can function in the improvement of phosphorus usage efficiency in wheat.