Optimization of the production of antifungal lipopeptide, iturin A, by B. subtilis was carried out in submerged cultural fermentation. In a shake-flask experiment, response surface methodology (RSM) was employed to optimize the cultivation conditions of Bacillus subtilis S3 for the enhancement of iturin A production. The optimal conditions for iturin A production obtained from RSM were pH 6.0, 0.93% maltodextrin, 1.11% glucose, 0.72% corn steep powder (C.S.P), 1.5 mM MgSO4, 0.75 mM KH2PO4, rotation speed 180 rpm and area of aeration 4.35 cm2. 57% increase of iturin A productivity (from 47.19 mg/L to 74.22 mg/L) was observed using the one-factor-at -a-time technique; however, 180% increase of iturin A productivity (from 47.19 mg/L to 132.23 mg/L) was observed with RSM. The iturin A production was further studied in a 5-L fermentor with a variation of agitation speed, aeration and baffles.In the 5-L fermentor, it was found that increased agitation speed improved the growth of B. subtilis and the production of iturin A. Aeration at 2 vvm gave excellent production of iturin A (175.52 mg/L) at 6 d of fermentation. The addition of baffles in the fermentor has significantly influenced the oxygen mass transfer coefficient (KLa) and iturin A production.