Four new steroidal saponins, named ophiopogonin P-S (1-4), together with eleven known ones (5-15) were isolated from the tuberous roots of Ophiopogon japonicus. Their structures were elucidated by spectroscopic and chemical analysis. Compounds 2-15 were evaluated for their cytotoxic activity against five human tumor cell lines (HepG2, HLE, BEL7402, BEL7403 and Hela). Compounds 2, 5, 6, 8 and 9 were cytotoxic for all cell lines tested. Compounds 7, 11 and 15 showed selective cytotoxicity against some of the cell lines. The structure-activity relationship of these compounds was discussed.