Extractive cultivation using polyethylene glycol (PEG)/potassium phosphate (KPi) aqueous two-phase systems (ATPSs) to produce and separate subtilin from Bacillus subtilis ATCC 6633 was investigated. B. subtilis ATCC 6633 cells grew in ATPS media, although growth rates were influenced by KPi concentration in the bottom phase in which cells are partitioned. In the ATPS media, the productivity and the stability of subtilin were increased and the release of subtilin was enhanced. Subtilin recovery from the top phase was 1.6-fold that in Spizizen minimal salts single-phase medium. The application of ATPS to extractive bioconversion is found to be possible in PEG/KPi systems which have high selectivity for the separation of the hydrophobic bioproducts.