The effect of different carbon sources, initial pH, and addition of divalent metal cations to the shaker flask cultivation medium was observed with the aim of improving iturin A production from Bacillus amyloliquefaciens B128. Among different C-sources tested, soft sugar enhanced iturin A production to 68.78 mg/L, which is 5 fold higher than nutrient broth cultivation concentration. This concentration further enhanced to 71.34 mg/L, after 6 days cultivation by the application of starting cultivation pH at 6.64. Furthermore, when this medium was supplemented with 0.2 mM of iron, iturin A concentration increased to 92.78 mg/L, after 5 days with no pH control cultivation. Interestingly, with the stating pH at 6.64 and 0.2 mM of ferrous sulfate, the highest iturin A production of 121.28 mg/L was obtained. Thus, the present study provides useful information for enhancing iturin A production in cultivation.