Resting cells of Pseudonocardia sp. KCTC 1029BP were used for the bioconversion of vitamin D-3 to calcifediol which is widely used to treat osteomalacia and is industrially produced by chemical synthesis. To obtain the maximum bioconversion yield of calcifediol by the microbial conversion of vitamin D-3, a two-step optimization process was used, including the Plackett-Burman and the central composite designs. Six variables, namely agitation speed, aeration rate, resting cell concentration, vitamin D-3 concentration, temperature, and pH, were monitored. Of these, aeration rate, resting cell concentration, and temperature were selected as key variables for calcifediol production and were optimized using the central composite design. Optimal bioconversion conditions obtained were as follows: aeration rate of 0.2 vvm, resting cell concentration of 4.7 % w/v, and temperature of 33 A degrees C. Using the optimal conditions, 356 mg calcifediol l(-1) was obtained with a bioconversion yield of 59.4 % in a 75 l fermentor. These are the highest values reported to date.