Four new compounds, aneglycoside A–C ( 1– 3) and timosaponin U ( 4), were isolated from the rhizomes of Anemarrhena asphodeloides. Their structures were determined through extensive spectroscopic analysis, chemical characteristics, and high-resolution mass spectrometry (HRMS). All the isolations were evaluated for cytotoxicity against HepG2, Hela, and SGC7901 human cancer lines. Compounds 1, 2, and 4 showed weak antiproliferative activities on HepG2, Hela, and SGC7901 cells.