A new lupine alkaloid, (-)-14β-hydroxymatrine (1), was isolated from the dry roots of Sophora tonkinensis together with 11 known lupine alkaloids. The absolute structure (5S, 6S, 7R, 11R, 14S) of the new base was confirmed by comparison of the natural product with a synthetic sample derived from (+)-matrine. This study reports the isolation of 12 lupine alkaloids from the roots of S. tonkinensis and the structural determination of the new alkaloid (1), including its absolute stereochemistry.