Phytochemical investigations from the roots of Cynanchum stauntonii led to obtain four new C_(21) steroidal glycosides(1–4) and one known compound stauntoside F(5). Their chemical structures were characterized by sophisticated analyses of IR, HRESI-TOF-MS, 1D, and 2D-NMR data, together with chemical methods, which showed interesting 13,14:14,15-disecopregnane-type skeleton or 14,15-secopregnane-type skeleton C_(21) steroidal glycosides. Among them, compound 1 was determined to be glaucogenin C 3-O-b-D-glucopyranosyl-(1 → 4)-b-D-cymaropyranosyl-(1 → 4)-b-D-digitoxopyranosyl-(1 → 4)-b-D-thevetopyranoside. Compound 2 was characterized to be hirundigenin 3-O-a-L-diginopyranosyl-(1 → 4)-b-D-cymaropyranosyl-(1 → 4)-b-D-digitoxopyranosyl-(1 → 4)-b-D-30-demethyl-thevetopyranoside. Compound 3 was identified to be(14S,16 S,20R)-14,16-14,20-15,20-triepoxy-14,15-secopregn-5-en-3-ol-3-O-a-L-cymaropyranosyl-(1 → 4)-b-D-digitoxopyranosyl-(1 → 4)-b-D-oleandropyranoside.Compound 4 was identified to be(14S,16 S,20R)-14,16-14,20-15,20-triepoxy-14,15-secopregn-5-en-3-ol-3-O-a-L-cymaropyranosyl-(1 → 4)-b-D-cymaropyranosyl-(1 → 4)-b-D-digitoxopyranosyl-(1 → 4)-b-Dthevetopyranoside. Among them, compound 2 was hirundigenin type C21 steroidal glycoside that existed in nature as epimers due to the presence of 14-hemiketal hydroxyl group in its structure. In addition, the anti-inflammatory and cardiomyocyte protective effects of compounds 1–4 were evaluated. We found that they exhibited significant protective effects on hypoxia/reoxygenation induced cardiomyocyte injury, but did not showed obvious anti-inflammatory function. Phytochemical investigations from the roots of Cynanchum stauntonii led to obtain four new C 21 steroidal glycosides (1-4) and one known compound stauntoside F (5). Their chemical structures were characterized by sophisticated analyzes of IR, HRESI-TOF-MS , 1D, and 2D-NMR data, together with chemical methods, which is interesting 13,14: 14,15-disecopregnane-type skeleton or 14,15-secopregnane-type skeleton C 21 steroidal glycosides. was determined to be glaucogenin C 3 -ObD-glucopyranosyl- (1 → 4) -bD-cymaropyranosyl- (1 → 4) -bD-digitoxopyranosyl- (1 → 4) -bD- thevetopyranoside. Compound 2 was characterized to be hirundigenin 3 (1 → 4) -bD-digitoxopyranosyl- (1 → 4) -bD-30-demethyl-thevetopyranoside. Compound 3 was identified to be (14S, 16 S , 20R) -14,16-14,20-15,20-triepoxy-14,15-secopregn-5-en-3-ol-3-OaL- cymaropyranosyl- (1 → 4) -bD-digitoxopyranosyl- → 4) -bD-oleandropyranoside. Compound 4 was Identified to be (14S, 16S, 20R) -14,16-14,20-15,20-triepoxy-14,15-secopregn-5-en-3-ol-3-OaL-cymaropyranosyl- ) -bD-cymaropyranosyl- (1 → 4) -bD-digitoxopyranosyl- (1 → 4) -b-Dthevetopyranoside. Among them, compound 2 was hirundigenin type C21 steroidal glycoside that existed in nature as epimers due to the presence of 14- In addition, the anti-inflammatory and cardiomyocyte protective effects of compounds 1-4 were evaluated. We found that they have significant protective effects on hypoxia / reoxygenation induced cardiomyocyte injury, but did not showed obvious anti-inflammatory function.