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Aims: Strains of Clostridium butyricum have been increasingly used as probiotics for both animals and humans. The aim of this study was to develop a growth medium for cultivating C. butyricum ZJUCB using a statistical methodology. Methods and Results: Response surface methodology (RSM) was used to evaluate the effects of variables, namely the concentrations of the glucose, pectin, soyabean cake extract, casein, corn steep flour, ammonium sulphate, sodium bicarbonate and the medium initial pH. A fractional factorial design was applied to study the main factors that affected the growth of a probiotic strain of C. butyricum currently preserved in our lab and the central composite experimental design was adopted to derive a statistical model for optimizing the composition of the fermentation medium. The experimental results showed that the optimum fermentation medium for the growth of C. butyricum was composed of 2% glucose (w/v), 0AE5% pectin (w/v), 0AE2% casein (w/v), 3AE98% soyabean cake extract, 0AE1% (NH 4 ) 2 SO 4 (w/v), 0AE124% NaHCO 3 (w/v), 0AE37% corn steep flour (w/v), 0AE02% MnSO 4 H 2 O (w/v), 0AE02% MgSO 4 7H 2 O (w/v) and 0AE002% CaCl 2 (w/v) at pH 7AE5. Conclusions: After incubating 24 h in the optimum fermentation medium, the populations of the viable organisms were estimated to be 10 9 CFU ml )1 . In the present study, we report the optimization of a growth medium that produced increased yields using statistical approach. Significance and Impact of the Study: The use of bacteria as a probiotic is showing increasing potential. The development of a growth medium that has a high yield is an obvious need, and the approach to optimizing a growth medium is innovative.