Four steroidal ketones were isolated from the holdfast of cultivated Laminaria japonica. The structures were characterized as ergosta-4,24(28)-diene-3-one (1), ergosta-4,24(28)-diene-3,6-dione (2), stigmasta-4,24(28)-diene-3-one (3) and stigmasta-4,24(28)-diene-3,6-dione (4), by spectral data. Compounds 2 and 4 were shown to be cytotoxic against the MCF-7 human breast cancer cell, and the growth of MCF-7 was inhibited by 96% and 79%, resp., at 10 μg/mL. It is the first report on the isolation of cytotoxic steroidal ketones from the kelp in the genus of Laminaria.