We studied the alkaloid constituents and bioactivity of Sophora flavescens (Leguminosae). The constituents were isolated with silica gel column chromatography, semi-preparative HPLC, Sephadex LH-20, and MPLC packed with MCI gel, and their structures were elucidated on the basis of physical characteristics and spectral data. Compounds 1 and 5-8 were evaluated for their in vitro cytotoxicity against human tumor HL-60, SMMC-7721, A-549, MCF-7, and SW-480 cell lines. Ten alkaloids were obtained, and their structures were identified as 17β-hydroxysophoridine (1), matrine (2), sophocarpine (3), sophoridine (4), isomatrine (5), 7,11-dehydromatrine (6), mamanine (7), sophoranol (8), oxymatrine (9), and oxysophocarpine (10). Compound 1 is a new alkaloid, and compound 7 was isolated from the Sophora flavescens for the first time. None of the compounds were cytotoxic to five human cancer cell lines. © 2014 Springer Science+Business Media.