Fed-batch fermentation was conducted to overproduce human lipocortin-1 (LC1) in recombinant Saccharomyces cerevisiae controlled by the GAL10 promoter. To optimize the feeding strategy, various fed-batch culture modes were performed with concentrated feed media containing carbon mixtures of galactose (Gal) and glucose (Glu) at three different concentration ratios (Gal:Glu ratios): 9:1, 1:1, 1:9. The cell growth, expression level of LC1, and the plasmid stability were investigated under these fed-batch culture modes, While both glucose and galactose were being fed to the fermenter, the glucose concentration in the culture broth was kept below 1 g/l for efficient gene expression. High cell concentrations of greater than 100 g dry cell weight/l mere achieved with these fed-batch culture modes. A significant amount of intact LC1 was found to be secreted into the culture medium, but proteolytically cleaved products (des1-26-LC1) were also observed in the culture medium. The fed-batch fermentation with the feed medium at a Gal: Glu ratio of 1:1 resulted in the highest LC1 total (intact LC1+des1-26-LC1) concentration of 500 mg/l, which corresponded to 1.73- and 1.83-fold increases over that produced with the media at Gal: Glu ratios of 1:9 and 9:1, respectively.