To investigate the chemical constituents from the root of Lasianthus acuminatissimus of Rubiaceae. Normal, reverse phase silica gel, macroporous resin column and HPLC chromatography were used for isolation. Spectroscopic methods (13C-NMR, 1H-NMR, DEPT, EI-MS) were used for identification. Six compounds were isolated and elucidated as isoscopletin(I), isoscopletin-6-O-beta-D-glucopyranoside (II), beta configuration of Mudanoside-A (Ill), asperuloside (IV), (-)-pinoresinol 4-O-beta-D-glucopyranoside (V), daucosterol (VI). These compounds were obtained from this plant for the first time.