A novel steroid, 1alpha,3beta,5beta-trihydroxy-24-methylenecholestan-6-one (gibberoketosterol) (1), along with four known steroids, was isolated from the lipophilic extracts of a Taiwanese soft coral Sinularia gibberosa. The structure of the new metabolite was determined on the basis of extensive spectral analyses and chemical reaction. The relative stereochemistry of gibberoketosterol was established by the NOESY experiments and analysis of the pyridine-induced deshielding effect of the axial hydroxy groups. Gibberoketosterol is the first example of 1alpha,3beta,5beta-trihydroxy-6-oxosteroids isolated from natural sources and was found to exhibit a moderate cytotoxicity against the growth of Hepa59T/VGH cancer cells.