The roots of Cynanchum wallichii Wight is rich in C21 steroidal glycosides according to previous research. In this study, a systematic chemical study was performed on the roots of C. wallichii, leading to the isolation of one previously undescribed C21 steroidal glycoside compound (1) and a known compound, gagamine (2). In vitro bioassay indicated that compound 1 showed a strong inhibitory effect on AGS cells, with the IC50 value 7.8 μM.