An enzymatic regeneration system consisting of laccase and the 2H+/2e− redox mediator Meldola’s blue (MB) was developed for the efficient oxidation of reduced nicotinamide adenine dinucleotide (NADH) cofactor and employed in the gram-scale oxidation of cholic acid (1) to its 7-keto derivative (1a) by 7α-hydroxysteroid dehydrogenase (7α-HSDH) in an aqueous, buffered reaction system. The regenerating enzyme, laccase, reduces molecular oxygen as terminal 4H+/4e− acceptor to water and concomitantly reoxidizes NADH via MB at high turnover rates. The regeneration system was successfully applied to quantitatively convert 1 (50 mM, 20.4 g) into 1a with a space-time yield of 5.8 mmol L−1 h−1. High total turnover numbers were achieved for 7α-HSDH (4.2×105) and laccase (1.1×106). Alternatively, the regeneration system was employed on the conversion of the methyl ester derivative of cholic acid (2, 200 mM, 5.9 g) dissolved in isopropyl acetate as organic solvent in a biphasic system. Due to the high concentration of 2 and the excellent performance of the enzymatic cascade reaction even under the harsh process conditions, space-time yields of up to 20 mmol L−1 h−1 (8.5 g L−1 h−1) of the produced 7-keto derivative 2a were obtained. The irreversibility and high driving force of the regenerating reaction allowed quantitative conversions and easy product recovery.