Abstract: OBJECTIVE To study the chemical constituents from the fruits of Vitex negundo L. var. cannabifolia. METHODS The chemical constituents were isolated and purified by chromatography on silica gel, ODS, MCI, Sephadex LH-20 columns, RP-HPLC and recrystallization. The structures were identified on the basis of physicochemical properties and spectral data analyses. RESULTS A total of 22 compounds were separated and identified from the 80% ethanol extract of Vitex negundo L. var. cannabifolia fruits, with 5 compounds from petroleum ether fraction and 17 compounds from dichloromethane fraction, including 9 lignans, 3 diterpenoids, 1 triterpenoid, 6 flavonoids and 3 phenolic acids compounds. CONCLUSION 4β-Paulownin, hinokiol and friedelin are isolated from this plant for the first time. 5,7,2',5'-Tetrahydroxyflavone is obtained from this genus for the first time.