Abstract: Response surface methodology was employed to optimize medium composition for the production of bacteriocin- like substance by Bacillus cereus XH25.The Plackett- Burmen design was used to evaluate the effects of different components in the culture medium.The initial p H of medium,soluble starch and( NH_4)_2SO_4 were found to significantly influence the bacteriocin- like substance production.The steepest ascent method was undertaken to access the optimal region of the above three significant factors,and then their concentrations were optimized by the central composite design and the response surface methodology. The results indicated that the optimized medium was initial pH of 6.15,soluble starch of 15.32 g / L and( NH_4)_2SO_4 of 6.02 g / L,and under this condition.Considering the cost,other insignificant factors were maintained at low levels: yeast powder of 5.00 g / L,sucrose of5.00 g / L,soybean meal of 5.00 g / L,MgSO_4 of 0.20 g / L and K_2HPO_4 of 2.00 g / L The predicted value 12.76 mm was verified subsequently by validation experiments value 12.92 mm. The excellent correlation between predicted and observed values further confirmed the adequacy and accuracy of the model obtained.And the antibacterial activity against Staphylococcus aureus ATCC 9144 was increased by 21.66% compared with the original medium,which provided a basis for further study on the large scale batch fermentation of production of bacteriocin- like substance by B.cereus XH25.