Cleavage of glucosinolates with myrosinase yields thioglycosidic compounds which have cancer chemoprevention activity. In this paper, glucosinolates in an extract (2.0 g) of broccoli seeds ( Brassica oleracea var. italica ) were separated by high-speed countercurrent chromatography (HSCCC) with the solvent system n -butanol-acetonitrile-10% ammonium sulfate solution 1:0.5:2.2 (v/v) to yield five glucosinolate compounds after desalting and decolorizing by MCI column chromatography. The five compounds, 7-methylsulfinylheptyl glucosinolate (22.4 mg), 4-pentenyl glucosinolate (33.6 mg), 3-butenyl glucosinolate (24.0 mg), 4-methylsulfinylbutyl glucosinolate (161.4 mg), and 3-methylsulfinylpropyl glucosinolate (29.6 mg), were identified by ESI-MS, 1 H NMR and 13 C NMR. The purity of the products was >98%, and 7-methylsulfinylheptyl glucosinolate was obtained from broccoli seeds for the first time.