Riboflavin producing lactic acid bacteria were isolated from fermented milk samples. Lactobacillus fermentum MTCC 8711, which produced higher amount of riboflavin, was used for the fermentative production of riboflavin. Response surface methodology with central composite design was used for the optimization of the fermentation medium for riboflavin production. Peptone, beef extract, glucose and K2HPO4 of the MRS broth were selected as variables. Among the tested variables, beef extract, glucose and K2HPO4 showed significant effect on the riboflavin production. Higher concentration of K2HPO4 exhibited positive response on riboflavin production. The validation run based on the optimum production in response surface plot resulted in the maximum (9.43 mg L-1) riboflavin yield, which was almost equivalent to the predicted value of 9.63 mg L-1. The Lb. fermentum strain produced four folds higher riboflavin in the optimized medium when compared to the unoptimized medium (2.28 mg L-1). © 2011.