Eleven C steroids were isolated from chloroform extract of roots of Cynanchum otophyllumby silica gel, MCI, ODS columns, and semi-preparative HPLC. Their structures were determined by spectroscopic data analysis as otophylloside B(1), caudatin-3-O-β-D-cymaropyranosyl-(1→4)- β-D-oleandropyranosyl-(1→4)-β- D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside(2), caudatin-3-O-β-D-oleandropyranosyl-(1→4)- β-D-oleandropyranosyl-(1→4)-β- D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside(3), caudatin-3-O-β-D-oleandropyranosyl- (1→4)-β-D-digitoxopyranosyl- (1→4)-β-D-cymaropyranoside(4), otophylloside O(5), gagamine-3-O-β-D-oleandropyranosyl- (1→4)-β-D-cymaropyranosyl- (1→4)-β-D-cymaropyranoside(6), sinomarinoside B(7), mucronatosides C(8), wallicoside J(9), stephanoside H(10), and qinyangshengenin-3-O-β-D-oleandropyranosyl- (1→4)-β-D-cymaropyranosyl-(1→4)- β-D-digitoxopyranoside(11). Among them, compounds 2-3, and 6-11 were separated from the roots of this plant for the first time.