The optimal conditions for High Pressure Carbon Dioxide (HPCD) inactivate of Bacillus subtilis spores were studied by response surface methodology (RSM).Box-Behnken design was applied to evaluate the effects of three indepent variables (pressure, temperature and pressure holding time) on the inactivation of Bacillus subtilis spores.A quadratic equation for HPCD inactivation was built.By analyzing the response surface plots and their corresponding plots as well as solving the quadratic equation, the experimental values were shown to be significantly in good agreement with predicted values since the adjusted determination coefficient (R2Adj) was 0.9862.