The chemical constituents in the ethanol extract from the root bark of Schisandra sphenanthera Rehder et E. H. Wilson were isolated and purified, and the in vitro antitumor activities of the compounds were compared. The results show that a total of eight compounds are isolated and identified, namely (-)-secoisolariciresinol-9′-O-β-D-glucopyranoside (1), schizanbuynin B (2), sphenlignan B (3), threo-(7R,8R)-guaiacyl-glycerol-β-O-4′-dihydroconiferyl ether (4), 3′,4-O-dimethylcedrusin (5), (±)-erythro-guaiacyl-glycerol-β-O-4′-dihydroconiferyl ether (6), dihydrodehydrodiconiferyl alcohol-4′-O-β-D-glucoside (7), and 3-hydroxymethyl-5-(γ-hydroxy-n-propyl)-2-(3′-methoxy-4′-O-α-rhamnopyranosylphenyl)-2,3-dihydrobenzofuran (8). All these compounds are isolated from S. sphenanthera for the first time. Among which, compounds 3, 6, and 8 all exhibit certain in vitro inhibitory activities against tumor cells, but only compound 6 shows better inhibitory activity against H460 cells compared with cisplatin (positive control).