Abstract: The chemical constituents of Dioscorea opposite Thunb. were isolated and purified by Diaion HP-20, Sephadex LH-20, Toyopearl HW-40, MCI Gel CHP-20, ODS, silica gel column chromatography and semi-preparative-HPLC. Nine compounds were found and their structures were elucidated by spectral data and physicochemical properties, which were identified as 2-(1',2',3'-trihydroxybutyl-4'-O-α-D-glucopyranoside)-6-(2",3",4"-trihydroxybutyl)-pyrazine (1), uracil (2), xanthine (3), hypoxanthine (4), thymine (5), adenosine (6), uridine (7), inosine (8), and 5'-deoxy-5'-methylsulphinyladenosine (9). The compound 1 is a new pyrazine derivative, and the compound 3-5, 8, 9 are found in this plant for the first time.