Human peptide transporter PepT2 contains 729 amino acids, 12-transmembrane domains. Essentially dipeptides, tripeptides and the similar peptide-like drugs can be transport by hPepT2 which plays an important role in nutrition and pharmacology. The fragment hPepT2(579-664) is located in the currently unknown function 10-12TMDs of hPepT2, which possibly contributes to important uptake and metabolism in vivo. So the BL21(DE3)-pET30a-hPepT2(579-664) the expression system was constructed, and the fragment protein was expressed under optimal condition, 300μM IPTG at 37 °C inducing 4 hours, and purified using Sephadex G-75 column to facilitate the further study on the structure and function of hPepT2.