In order to delay the sporulation and improve the growth efficiency of Clostridium butyricum, the effects of guanine (0.2 g/L) and Ile (0.5 g/L) on the growth and acids metabolism of C. butyricum in 20 L bioreactor were investigated. The results showed that the maximum biomass with the addition of guanine or Ile reached 5.31×108 CFU/mL and 4.65×108 CFU/mL, respectively, which was increased by 22.91% and 7.64% compared with the control (4.32×108 CFU/mL). The coefficient of butyric acid to maximum biomass (Yp/x) was decreased by 48.64% and 40.54% compared with the control (0.37 g/g), respectively. Gunanine and/or Ile addition could reduce the specific butyrate productivity, enhance the growth efficiency and delay the sporulation of C. butyricum. The biomass reached 5.4×108 CFU/mL when guanine (0.2 g/L) and Ile (0.5 g/L) were simultaneously added to the fermentation medium, which was 25% higher than that of the control. This research provides new methods for industrial fermentation of C. butyricum.