The nutritional medium requirement for biomass and triterpenoid production by Antrodia cinnamomea AC0623 strain was optimized. Box-Behnken was applied to optimize biomass and triterpenoid production. According to response surface methodology (RSM), the optimum concentrations of N-source were determined. The results indicate that when a submerged culture in shake flasks was operated at 28 degrees C, initial pH 5.5, and rotation speed 105 rpm, the biomass and triterpenoid content in dry basis could be increased to 3.20% (w/w) and 31.8 mg/g, respectively. The experiments were further scaled up to 100- and 700-1 fermentors. Higher content of triterpenoids (63.0 mg/g) was obtained in 700-1 fermentations by means of the control of cultural conditions and the modification of medium composition based on the RSM.