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Two new and two known C21 steroids were isolated from Cynanchum atratum.All compounds showed obvious cytotoxic activities against HepG2 cells.One C21 steroids can induce apoptosis through a mitochondria-mediated pathway and cell cycle arrest in the HepG2 cells.Two new (1–2) and two known C21 steroids (3–4) were isolated from the roots of Cynanchum atratum. Their structures were elucidated by detailed 1D and 2D spectroscopic. The MTT assay showed that compounds 1–4 displayed obvious cytotoxic activities against HepG2 cells with IC50 values ranging from 10.19 μM to 76.12 μM. Compounds 1–3 also exhibited cytotoxic effects in A549 cells with IC50 values of 30.87–95.39 μM. Compound 3 showed the antiproliferative activity via G0/G1 cell cycle arrest and proapoptosis in HepG2 cells by Flowcytometry analysis. Western blotting analysis revealed that compound 3 could induce HepG2 cell apoptosis via the mitochondrial pathway by downregulating Bcl-2 expression, upregulating Bax protein expression, and activating caspase-9 and caspase-3.