Two new steroids, vladimuliecins A (1) and B (2), were isolated by bioassay-guided fractionation from the rhizome of Vladimiria muliensis. Compounds 1 and 2 are the first examples possessing a pentacyclic 3alpha,5alpha-cyclopregnane-type framework. The structures of vladimuliecins A (1) and B (2) and their deacetylated derivative (3) were determined on the basis of IR, MS, 1D NMR, 2D NMR, and X-ray data analyses. The absolute configuration of the 10 stereogenic centers of compounds 1 and 2 was determined to be 3R,5R,6R,8S,9R,10R,13R,14R,17S,20R by means of auxiliary chiral MTPA derivatives and optical rotation calculation. A probable biosynthesis pathway to 1 and 2 is also proposed and discussed. In addition, the cytotoxicity of compounds 1, 2, and 3 was evaluated against selected cancer cell lines, including human leukemia cell (HL-60), human hepatoma cell (SMMC-7721), and human cervical carcinoma cell (HeLa) lines.