We report the very efficient biotransformation of cholic acid to 7-keto- and 7,12-diketocholic acids with Acinetobacter calcoaceticus lwoffii. The enzymes responsible of the biotransformation (i.e. 7α- and 12α-hydroxysteroid dehydrogenases) are partially purified and employed in a new chemo-enzymatic synthesis of ursodeoxycholic acid starting from cholic acid. The first step is the 12α-HSDH-mediated total oxidation of sodium cholate followed by the Wolf–Kishner reduction of the carbonyl group to chenodeoxycholic acid. This acid is then quantitatively oxidized with 7α-HSDH to 7-ketochenodeoxycholic acid, that was chemically reduced to ursodeoxycholic acid (70% overall yield).