Two new (1–2) and three known (3–5) C21 steroidal glycosides were isolated from Cynanchum stauntonii. Their structures were elucidated on the basis of 1D and 2D-NMR spectroscopic data as well as HRTOFMS analysis. The cytotoxicity of the compounds against A549, HepG2, and 4T1 cell lines were evaluated by MTT assay. Compound 4 exhibited good inhibitory activities with the IC50 values 26.82, 12.24, and 44.12 μM, respectively. Furthermore, compound 4 could induce G1 phase arrest, upregulate the expression levels of caspases-3, -9, and Bax, and downregulate the expression level of Bcl-2. These results indicated that compound 4 might be valuable to anticancer drug candidates.