Isomaltulose is a structural isomer of sucrose commercially used in food industries. Glucosyltransferase produced by Erwinia sp. D12 catalyses an intramolecular transglucosylation of sucrose giving isomaltulose. The Experimental design and response surface methodology were applied for the optimization of the nutrient concentration in the culture medium for the enzyme production in shaken flasks at 200 rpm and 30ºC. The three variables involved in this study were sugar cane molasses, bacteriological peptone and yeast extract Prodex Lac SD®. The statistical analysis of the results showed that, in the range studied, all the factors had a significant effect (p < 0.05) on glucosyltransferase production and the highest enzyme activity was observed in culture medium containing sugar cane molasses (160 g/L), bacteriological peptone (20 g/L) and yeast extract Prodex Lac SD® (15 g/L). Maximum glucosyltransferase activity of 29.88 U/mL was achieved in a 6.6-L fermenter using the optimized medium. Free Erwinia sp. D12 cells were used for isomaltulose production from sucrose during fifteen successive batches. The final isomaltulose concentration of 75.6% obtained in the first batch increased to 77.21% (mean value) in the other fourteen batches and the productivity of 1.1 g/L x hr was obtained in batch process.