Chemotaxis of sperm toward eggs during fertilization is a crucial event for species conservation, particularly for animals living in aquatic environments. Relevant chemical attractants have been found from a few marine organisms such as sea urchins and corals. We previously showed that sperm of the ascidian Ciona intestinalis is activated and then attracted toward the egg by a factor released from the egg, which led to the purification of sperm-activating and attracting factor (SAAF) from the egg-conditioning medium of Ciona intestinalis. The structure of SAAF was proposed to be a novel polyhydroxysterol sulfate (1) on the basis of 2D-^1H NMR and FAB-MS/MS analysis using no more than approximately 4μg of a specimen. However, there still remained small but serious ambiguity that the biological activity might be attributed to a minor constituent. Synthesis is, therefore, essential for the unequivocal identification of 1 as the active principle, and for the complete structure elucidation including the stereochemistry at C25. SAAF and its C-25 epimer were synthesized from chenodeoxycholic acid (8) in 19 steps via the pivotal intermediate 6 and both the sperm-activating and attracting activity of them were bioassayed. Synthetic SAAF activated the sperms of ascidian Ciona intestinalis at 3.7nM and concurrently exhibited the attracting activity at <10nM, which led to the unambiguous structure determination of SAAF and the verification of its dual activity. It is noteworthy that 25-epi-SAAF turned out to possess the comparative activities as those of SAAF.