Phytochemical investigation on the whole plant of Ypsilandra thibetica led to the isolation of five new cholestane derivatives. The structures of 1–5 were elucidated by comprehensive spectroscopic analyses of their data and chemical methods. The absolute configurations of compounds 1–3 were ambiguously assigned by single crystal X-ray diffraction analysis. Compound 1 is the first cholestane glycoside with two oxygen bridges between C-16/C-23 and C-18/C-23. Compound 2 is the first 23-spirocholestane derivative with a six-membered ring between C-24/C-23 of the aglycone moiety and C-1′/C-2′ of the D-arabinose ligand, while compounds 3–5 are new 23-spirocholestane derivatives with a D-fucose ligand. The cytotoxic, anti-inflammatory, and anti-angiogenic activities of these isolated compounds were evaluated. Compound 5 showed anti-angiogenic activity in zebrafish embryos.