New marine steroids, aragusterol A (1), B (2) and C (3), were isolated from the Okinawan sponge of the genus, Xestospongia. Aragusterol A and C strongly inhibited the cell proliferation of tumor cells in vitro, and also showed potent in vivo antitumor activity toward L1210 in mice. The planar structure of aragusterol A (1) was elucidated based on spectroscopic analysis and chemical reaction (Scheme I). PCC oxidation of 1 gave 4,5 and 6. Zn-AcOH reduction of 6 gave 7 which was shown to be identical with 7 obtained from hecogenin acetate (8), elucidating the planar structure of the nucleus as well as the stereochemistry of the nucleus except for C-12. The structure of the side chain was determined by formation of 12 on treating 10 with H_5IO_6 followed by NaBH_4 and then p-nitrobenzoyl chloride. The stereochemistry at C-12 was determined based on the coupling constant of H-12 and observation of NOE between H-12 and H-14 in 1. The 22R configuration was elucidated by applying modified Mosher's method, and the stereochemistry at C-20 was determined based on observation of NOE between H-21 and H-22 in 11. The stereochemistry at C-24, 25 and 26 was established by the synthesis of 12 and 23 as shown in Scheme II and III. The structure of aragusterol B was shown to be 2 except for the stereochemistry at C-20 on the basis of spectroscopic analysis and chemical reaction (Scheme IV). The S configuration at C-20 was determined by X-ray crystallographic analysis. The structure of aragusterol C (3) was also established by X-ray crystallographic analysis.