The reaction conditions for the nicotinamide production by Rhodococcus erythropolis MTCC 1526 have been optimized by statistical experimental design. Application of this approach in the bioprocess can result in rallied product yield, reduced development time, and process variability. In this investigation, response surface methodology and central composite design were employed to predict the levels of variables such as reaction pH ( 6.5, 7, and 7.5), temperature ( 15, 20, and 25 C), cell concentration ( 190, 200, and 210 mg/ml), and substrate concentration ( 18, 20, and 22 mM) on the production of nicotinamide. A total of 22 experiments were carried out in shake flasks, and a three-dimensional response surface was generated to determine the effect of crucial reaction parameters for the maximum conversion of 3-cynopyridine to nicotinamide. Using this methodology, the optimal values for the reaction conditions were reaction pH 6.85, temperature of 24.8 degrees C, cell concentration of 190.98 mg/ml, and substrate concentration of 21.98 mM. This statistical approach led to the increase of conversion of 3-cynopyridine (93%) as compared to the conversion obtained by one-factor-at-a-time approach (84%).