The specific oxidation of 12α-OH group of hydroxysteroids is required for the preparation of cheno- and ursodeoxycholic acid (CDCA and UDCA, respectively). The C12 oxidation of hydroxysteroids into their 12-oxo derivatives can selectively be performed by employing 12α-hydroxysteroid dehydrogenases. These enzymes use NAD(P)+ as an electron acceptor, which has to be re-oxidized in a so-called “regeneration system”. Recently, the enzyme NAD(P)H oxidase (NOX) was applied for the regeneration of NAD+ in the enzymatic preparation of 12-oxo-CDCA from cholic acid (CA), which allows air to be used as an oxidant. However, the NOX system suffers from low activity and low stability. Moreover, the substrate loading is limited to 10 mM. In this study, the laccase/mediator system was investigated as a possible alternative to NOX, employing air as an oxidant. The laccase/mediator system shows higher productivity and scalability than the NOX system. This was proven with a preparative biotransformation of 20 g of CA into 12-oxo-CDCA (92% isolated yield) by employing a substrate loading of 120 mM (corresponding to 50 g/L). Additionally, the performance of the laccase/mediator system was compared with a classical ADH/acetone regeneration system and with other regeneration systems reported in literature.