Eight new C21 steroidal glycosides, namely cynanotins A-H (1-8), together with fifteen known analogues, were isolated from the roots of Cynanchum otophyllum. Their structures were elucidated by spectroscopic analysis and chemical methods. In this study, all of isolates were tested for their vitro inhibitory activities against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW480). Compounds 3-15 showed moderate cytotoxic activities against HL-60 cell lines with IC50 values ranging from 11.4 to 37.9 mu M. Compounds 5, 9, and 10 showed marked or moderate cytotoxic activities against five human tumor cell lines with IC50 values ranging from 11.4 to 36.7 mu M. Compound 11 displayed moderate cytotoxic activities against HL-60, SMMC-7721, MCF-7 and SW480 cell lines with IC50 values of 12.2-30.8 mu M. Compared to the positive control (IC50: 35.0 mu M), compounds 5, 9-11 exhibited more potential inhibitory activity against MCF-7 cells (IC50: 16.1-25.6 mu M). (C) 2018 Published by Elsevier Ltd.