Response surface methodology (RSM) was used to optimize sporulation medium components for improving spores concentration in I. lacteus fermentation broth. Five factors of sporulation medium components, glucose (X1), peptone (X2), yeast extract (X3) MgSO4·7H2O (X4) and VB1 (X5), were chosen to optimize through a five-level, five-factor central composite design (CCD) base on single factor experiments. The concentration of medium components which influenced the sporulation of I. lacteus was further optimized using RSM. The results show that The medium that consists of 4.15 g/L peptone, 2.04 g/L yeast extract, 5.10 g/L glucose, 0.17 g/L MgSO4·7H2O and 0.03 g/L VB1 enhance spores concentration significantly . The final spores concentration (2.19×105 /mL) by using the optimized medium was about five times higher than normal medium.