Four new C21 steroidal glycosides, named otophylloside T (2), U (3), V (4) and W (10), together with six known compounds including beishouwubenzophenone (1), the enzymatic hydrolysate of wallicoside (5), Otophylloside M (6), Cynanauriculoside E (7), Wallicoside (8) and Cynauricuoside C (9), were isolated from the roots of Cynanchum otophyllum Schneid. The structures of the four new C21 steroidal glycosides were elucidated as qingyangshengenin-3-O-β-d-thevetopyranosyl-(1→4)-β-d-cymaropyranosyl-(1→4)-β-d-digitoxopyranoside (2), qingyangshengenin-3-O-β-d-thevetopyranosyl-(1→4)-β-d-cymaropyranosyl-(1→4)-β-d-cymaropyranoside (3), caudatin-3-O-β-d-glucopyranosyl-(1→4)-β-d-oleandropyranosyl-(1→4)-β-d-cymaropyranosyl-(1→4)-β-d-digitoxopyranoside (4) and caudatin-3-O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranosyl-(1→4)-β-d-oleandropyranosyl-(1→4)-β-d-oleandropyranosyl-(1→4)-β-d-cymaropyranosyl-(1→4)-β-d-cymaropyranoside (10) by extensive spectroscopic evidence.