Two new (1-2) and two known C-21 steroids (3-4) were isolated from the roots of Cynanchum atratum. Their structures were elucidated by detailed 1D and 2D spectroscopic. The MIT assay showed that compounds 1-4 displayed obvious cytotoxic activities against HepG2 cells with IC50 values ranging from 10.19 mu M to 76.12 mu M. Compounds 1-3 also exhibited cytotoxic effects in A549 cells with IC50 values of 30.87-95.39 mu M. Compound 3 showed the antiproliferative activity via GO/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.