A method for the synthesis of 3alpha,7alpha,14alpha-trihydroxy-5beta-cholan-24-oic acid which is a possible candidate of bile acid metabolite in vertebrates was developed. The principal reactions involved were 1). stereoselective remote-hydroxylation of methyl ursodeoxycholate diacetate with dimethyldioxirane, 2). site-selective protection at C-3 by tert-butyldimethylsilylation of the resulting 3alpha,7alpha,14alpha-trihydroxy ester, 3). oxidation of the diol with pyridinium dichromate adsorbed on activated alumina, 4). stereoselective reduction of the 7-ketone with zinc borohydride, and 5). cleavage of the protecting group at C-3 with p-toluenesulfonic acid. A facile elimination of the 14alpha-hydroxy group under an acidic or neutral condition is also described. The synthetic reference compound is now available for comparison with unidentified biliary bile acids detected in vertebrate bile.