Prodigiosin, a pigment produced by Serratia marcescensinhibits the growth of different microorganisms and the proliferation of some human cancer cell lines. Prodigiosin is usually produced by fermentations of substrates such as starch and proteins, andpig-ment yield depends on the concentration of the carbon source, stirring speed of cultures, temperature and time of incubation,nitro-gen sources, and pH of medium. In the present study, pigment production was assessed using two types of casein as substrate; pigment yield was greater when casein precipitated with vinegar was used as substrate than when high purity casein was. The maxi-mum prodigiosin production was achieved with 10 g/L casein at pH 8.0. Additionally, supplementation of culture media with glu-cose was found to considerably decrease prodigiosin production and growth inhibition of Staphylococcus aureus, which is directly related to pigment yield. Production in stirred-tank bioreactor at 0.75 vvm aeration was higher than that at 0.5 and 1.0 vvm. Sub-strate type, concentration and pH affected pigment production in Erlenmeyer flasks, whereas aeration rate influenced pigment pro-duction in a stirred-tank bioreactor.