Clostridium cellulolyticum is an important strain capable of producing cellusome for lignocellulose degradation.However, this strain is difficult to cultivate and reach high cell density.In this study, culture conditions for high cell density culture of Clostridium cellulolyticum was optimized using response surface methodology in this study.First, Plackett-Burman design was employed to reveal the influential factors for C.cellulolyticum growth rate, which were determined as yeast extract concentration, cellobiose concentration and culture temperature.Next, the steepest ascent path design was applied for approximation to the optimal culture conditions for obtaining high cell density.At last, the central composite design and the response surface analysis were used to determine the optimal levels of the influential factors, which were 3 g/L, 7 g/L and 34 ℃, for yeast extract concentration, cellobiose concentration and culture temperature, respectively.The optimized culture medium was applied for flask culture of C.cellulolyticum, and raised the cell concentration from 0.303 to 0.586 in terms of OD600, with an increase of 93.4%.In a fermentor with controlled pH for batch culture, the biomass concentration reached an OD600 of 3.432, which was 2.8 times higher than the value ever reported so far.These results provide the basis for the further study on C.cellulolyticum culture techniques and applications.