Abstract: Two new alkaloids along with eleven known ones, were isolated from the 70% ethanol extract of the roots of Scrophularia ningpoensis Hemsl. by various chromatographic methods, including silica gel, Sephadex LH-20, reverse phase C₁₈ and semi-preparative HPLC. Their structures were determined by physicochemical properties and spectroscopic methods, including UV, IR, MS, 1D/2D NMR and ECD. They were identified as 11α-isopropylcytisine (1), 13α-isopropylcytisine (2), uracil (3), (S)-5-hydroxypyrrolidin-2-one (4), pterolactam (5), methyl-L-pyroglutamate (6), kaempferide (7), luteolin (8), 5,6,7,4'-tetramethoxyflavone (9), 6'-hydroxy-2',3',4,4'-tetramethoxychalcone (10), scopolin (11), hymexelsin (12), and 8-hydroxycoumarin (13). Among them, compounds 1 and 2 are two new compounds, while compounds 3, 4, 7-12 were isolated from this plant for the first time. An antitumor activity assay showed that compounds 8 and 9 had weak cytotoxicity against breast cancer cells (4T1).