Twenty-three undescribed previously C steroidal glycosides, named saccatols D–K and cynsaccatols I−W, together with six known analogs were isolated from the roots of Cynanchum auriculatum Royle ex Wight (Apocynaceae). Their structures were elucidated by a variety of spectroscopic techniques, as well as HRESIMS and GC method. All isolated compounds were tested their neuroprotective effects on the model of HO-induced damage to PC12 cells. Except saccatols D–F, J, cynsaccatol V and wilfoside A, other twenty-three isolates produced significantly activities against HO-induced cell damage (P < 0.001). Furthermore, cynsaccatols I, N, O and S can obvious inhibition of damaged PC12 cell apoptosis at their dosages of 1 μM by Annexin V-FITC/PI double staining assay with flow cytometry.