Abstract: D-Xylose-U-¹³C₅ was synthesized with D-glucose-U-¹³C₆ as starting material via process of protection with double acetone, selective hydrolysis with acid, oxidation, reduction and hydrolysis with base. The total yield of D-Xylose-U-¹³C₅ was 38.6% base on D-glucose-U-¹³C₆. The target compound and their intermediates were analyzed and characterized by GC, GC-MS, HPLC and LC-MS, accordingly. The results showed that their isotopic abundance and purity could meet the required specification. The structure of final product was D-Xylose-U-¹³C₅, ¹³C abundance was 99.1%， and chemical purity was 99.9%, which were high enough for use in ¹³C breath test.