Three new steroidal sapogenins, 3fl,8fl,14fl-trihydroxycarda-5,20(22)-dienolide (1), (20R)-12fl,20-epoxy3fl,8fl,14fl-trihydroxy-17a-pregn-5-en-17-yl 4-hydroxy-3-methoxybenzoate (2), and 3,20-dioxo-8fl,14fl,17fltrihydroxy-17a-pregn-5-en-12fl-yl 4-hydroxybenzoate (3), along with four known C21-steroidal sapogenins (4-7) were isolated from the hydrolysate of Cynanchum otophyllum roots. Among them, compound 1 harbouring a rare fl-OH at the C-8 position in the cardenolide skeleton, was identified for the first time in the genus Cynanchum. Their structures were elucidated via extensive spectroscopic analysis, including UV, IR, HR-ESI-MS, 1D and 2D NMR. Compounds 1-7 were evaluated for their cytotoxic activities against four cancer cell lines (MCF-7, H1299, HeLa, and HepG2). Compound 1 exhibited significant inhibitory effects on the growth of the four cell lines, exhibiting IC50 values ranging from 6.86 to 22.09 mu M.