Six new steroidal saponins (1–6) and one known steroidal saponin (7) were obtained from the roots of Ophiopogon japonicus (L. f.) Ker-Gawl. Their structures were determined by the detailed analysis of extensive nuclear magnetic resonance and mass spectroscopic data. The in vitro cytotoxic activities of these compounds against MDA-MB-435, HepG2 and A549 cell lines were also investigated.