Plackett-Burman and Response Surface Methodology were used to optimize fermentation medium for enhancing butanol production by Clostridium beijerenckii strain CHTa. Optimization using Plackett-Burman design showed yeast extract, MgSO4.7H2O and glucose to have signiï¬cant impact over other factors, namely (NH4)2SO4, CaCO3, FeSO4.7H2O, cysteine hydrochloride, sodium thioglycolate and pH. To find out the optimal concentrations and interactions between the selected factors, further experiments were carried by applying Central Composite Design. The experimental results demonstrated glucose and MgSO4.7H2O as the key components over yeast extract responsible for production of butanol by strain CHTa. A mathematical model was established to predict the optimum values for factors. Under optimal conditions, practical butanol yield of 10.08 g L-1 and productivity of 0.45 g L-1 h-1 were obtained.