For the complete structure elucidation of an endogenous sperm-activating and -attracting factor isolated from eggs of the ascidian Ascidia sydneiensis ( Assydn-SAAF), its two possible diastereomers with respect to C-25 were synthesized. Starting from ergosterol, the characteristic steroid backbone was constructed by using an intramolecular pinacol coupling reaction and stereoselective reduction of a hydroxy ketone as key steps, and the side chain was introduced by Julia-Kocienski olefination. Comparison of the NMR data of the two diastereomers with those of the natural product led to the elucidation of the absolute configuration as 25 S; thus the complete structure was determined and the first synthesis of Assydn-SAAF was achieved.