Supplement of Fe2+ into fermentation medium was utilized as a tool to optimize the iron-mediated enhancement of surfactin production from Bacillus subtilis ATCC 21332. Up to 3000 mg L-1 of surfactin was produced using an iron-enriched minimal salt (MS) medium amended with an optimal Fe2+ dosage of 4.0 mM, leading to 8-fold and 10-fold increase in cell concentration and surfactin yield, respectively, as compared to those without Fe2+ supplement. In addition to resulting in an optimal production yield of surfactin, a supplement of 4.0 mM of Fe2+ also propelled maximum overall surfactin production rate to a highest value of 24 mg L-1 h(-1). Our results also show that production of surfactin followed a growth-associated kinetic model. The best yield coefficient estimated from the model was ca. 162 mg surfactin (g dry cell)(-1). The supernatant of the iron-enriched culture of B. subtilis ATCC 21332 exhibited the ability to emulsify kerosene and achieved a maximum emulsion index (E-24) of 80% for culture supplemented with 4.0 mM of Fe2+. Comparison of emulsion index and the corresponding surfactin production indicates that the emulsification activity was essentially contributed by surfactin.