To understand the chemical constituents from the acid catalytic hydrolysate of the root of Cynanchum otophyllum Schneid.Four lignan compounds were isolated from the acid catalytic hydrolysate of the 95% ethanol extract of the root of C. otophyllum by means of multiple columnchromatography techniques including silica gel,ODS and preparative HPLC.Their structures were elucidated by spectroscopic analysis and comparison of their spectroscopic data with those reported in the literature to be 9′-methoxynegundin B (1),(+)-isolariciresinol (2),dehydroconiferyl alcohol (3),5-methoxydehydroconiferyl alcohol (4).Among them,1 was new compound and compounds 2-4 were obtained for the first time in the genus Cynanchum.The cytotoxicities of compounds 1-4 toward four human carcinoma cell lines,HeLa,H1299,HepG2,and MCF-7 cells were evaluated by MTT method.The results showed that compounds 2 and 3 had strong inhibitory effects on the growth of HepG2 carcinoma cell line.