Five known compounds, cimigenol (1), 25-O-acetylcimigenol (2), cimigenol 3-O-β-D-xylopyranoside (3), ferulic acid methyl ester (4), and visnagin (5), were isolated from Cimicifugae Rhizoma (Ranunculaceae). The chemical structures of these compounds were determined on the basis of spectroscopic analyses including 2D NMR. Compounds 1-5 were evaluated for their cytotoxic activity against the HL-60, MCF-7, and A549 cancer cell lines in in vitro. Among them, compounds 4 and 5 showed moderate inhibitory activity against HL-60 cancer cell lines with IC50 values of 24.8 and 18.1 μM, respectively.