Clostridium butyricum, an anaerobic spore-forming bacillus, is a common human and animal gut commensal bacterium. Spore is an important structure for C. butyricum to tolerate environmental stress. However, it is not easy to form in common fermentation process of C. butyricum. In this study, the parameters for optimizing the spore formation of C. butyricum NN-2 were defined. The results showed that the pH value was a crucial factor that significantly affected the spore formation of C. butyricum NN-2. Down-regulation steps of pH value from 6.5 to 5.5 over time during the cultural process significantly (p 90%. In addition, the duration of pH regulation also had significant effects on the spore formation of C. butyricum NN-2. The results revealed a highly effective strategy for enhancing the spore production of C. butyricum.