Four new steroidal constituents ( 1– 4) along with two known steroidal glycosides ( 5 and 6) were isolated from the roots and rhizomes of Smilacina japonica. Analysis of their physicochemical properties and spectroscopic profiles identified the compounds as (25 S)-5 α-spirostan-9(11)-en-3β, 17 α-diol ( 1); (25 S)-5 α-spirostan-9(11)-en-3β, 12β-diol ( 2); (25 S)-5 α-spirostan-9(11)-en-3β, 17 α-diol-3- O-β- d-glucopyranoside ( 3); (25 S)-5 α-spirostan-9(11)-en-3β, 17 α-diol-3- O-β- d-glucopyranosyl-(1→2)-[β- d-glucopyranosyl-(1→3)]-β- d-galactopyranoside ( 4); japonicoside B ( 5); and japonicoside C ( 6). All six compounds showed cytotoxic activity against SMMC-7712, Bel-7402, A549, H460, and K562 human cancer cells.