Statistics-based experiment designs were used to optimize the culture medium (glucose, yeast extract, IPTG, tween-60, and CaCl2) for cutinase production by recombinant Escherichia coli. A 2^(5-1) fractional factorial design augmented with center points revealed that glucose, yeast extract, and IPTG were the most significant factors, whereas the other factors were not important within the levels tested. The method of steepest ascent was used to approach the proximity of optimum, followed by a central composite design to develop a response surface for culture condition optimization. The optimum culture medium for cutinase production was found to be: glucose 33. 92 g/L, yeast extract 30.92 g/L, and IPTG 0.76 g/L. A cutinase production of 145.27±1.5 U/mL, which was in agreement with the prediction, was observed in triplicate verification experiments. The results obtained here verified the effectiveness of the applied methodology and may be helpful for cutinase production on an industrial scale.