Spore-forming Bacillus sp. has been extensively studied for their probiotic properties. In this study, an acid-treated rice straw hydrolysate was used as carbon source to produce the spores of Bacillus coagulans. The results showed that this hydrolysate significantly improved the spore yield compared with other carbon sources such as glucose. Three significant medium components including rice straw hydrolysate, MnSO4 and yeast extract were screened by Plackett-Burman design. These significant variables were further optimized by response surface methodology (RSM). The optimal values of the medium components were rice straw hydolysate of 27% (v/v), MnSO4 of 0·78 g l-1 and yeast extract of 1·2 g l-1 . The optimized medium and RSM model for spore production were validated in a 5 l bioreactor. Overall, this sporulation medium containing acid-treated rice straw hydrolysate has a potential to be used in the production of B. coagulans spores.