By comprehensively applying various separation techniques such as silica gel, ODS, and Sephadex LH-20 column chromatography, as well as preparative HPLC, fourteen C21 steroidal compounds were isolated from the chloroform extract of Cynanchum wallichii Wight. The structures of these compounds were identified through spectroscopic techniques including mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR), ultraviolet spectroscopy (UV), and infrared spectroscopy (IR) methods as cynanotonin E (1), qingyangshengenin (2), qinyangshengenin-3-O-β-D-cymaropyranoside-(1→4)-β-D-cymaropyranoside (3), qinyangshengenin-3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosy-(1→4)-β-D-digitoxopyranoside (4), qinyangshengenin-3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-digitoxopyranoside (5), 12-O-benzoylsarcostin-3-O-β-D-oleandropyranosy-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl (6), 12-O-benzoylsarcostin-3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-digitoxopyranosyl (7), caudatin-3-O-β-D-digitoxopyranoside (8), caudatin-3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranoside (9), caudatin-3-O-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside (10), caudatin-3-O-β-D-cymaropyranosyl-(1→4)-β-D-digitoxopyranoside (11), otophylloside B (12), otophylloside E (13) and otophylloside F (14). Among them, compound 1 is a new compound. The cytotoxic activities of compounds 1-14 against human hepatocellular carcinoma HepG2 cells were tested using the MTS method. The results indicated that all compounds exhibited good inhibitory activities against the proliferation of HepG2 cells, with IC₅₀ values ranging from 4.3 ± 1.25 - 26.3 ± 1.65 μmol·L^-1. Among them, the inhibitory activities of compounds 1, 3 and 6-14 were all stronger than that of the positive control cisplatin (DDP, IC₅₀: 15.9 ± 1.25 μmol·L^-1).