Abstract Cytidine 5′-diphosphorylcholine (CDP-choline) was produced using a high efficiency ATP regeneration system and the Kennedy pathway in whole cells of Saccharomyces cerevisiae As 2.398. Out of eight variables, KH 2PO 4, glycerol and (NH 4) 2SO 4 were considered to be the most significant factors by response surface methodology including a Plackett–Burman design, path of steepest accent and central composite design. The optimum levels of the three variables were 20.13 g/L KH 2PO 4, 12.35 g/L glycerol and 0.49 g/L (NH 4) 2SO 4, respectively. Energy utilization efficiency increased from 10.59% to 16.72% and choline chloride conversion yields increased from 12.35% to 42.78%. A high efficiency ATP regeneration system improves CDP-choline production.