The twelve-protostane analogues were synthesized from alisol B 23-acetate and assessed for their in vitro antitumor activity against six different human and murine tumor cell lines. Of the compounds synthesized, 23S-acetoxy-24R(25)-epoxy-11$eta$,23S-dihydroxyprotost-13(17)-en-3-hy-droxyimine (12) exhibited significant cytotoxic activities against A549, SK-OV3, B16-F10, and HT1080 tumor cells with $ED_{50}/$ values of 10.0, 8.7 ,5.2, and 3.1 ${mu}g$/ml, respectively. Furthermore, 23S-acetoxy-13(17),24R(25)-diepoxy-11$eta$-hydroxyprotost-3-one (5), 13(17),24R(25)-diepoxy-11$eta$, 23S-dihydroxyprotostan-3-one (6), 24R,25-epoxy-11$eta$,23S-dihydroxyprotost-13(17)-en-3-one (7), and 11$eta$,23S,24R,25-tetrahydroxyprotost-13(17)-en-3-one (9) showed moderate cytotoxic activities against 816-F10 and HT1080 tumor cells. These results mean that a hydroxyimino group at C-3 position in the protostane-type terpene enhances cytotoxic activity.