L-Lactic acid production by Lactobacillus casei was used as a model to study the mechanism of substrate inhibition and the strategy for enhancing L-lactic acid production. It was found that the concentration of cell growth and L-lactate decreased with the increase of glucose concentration and fermentation temperature. To enhance the osmotic stress resistance of the strain at high temperature, a mutant G-03 was screened and selected with 360 g/L glucose at 45°C as the selective criterion. To further increase the cell growth for lactic acid production, 3 g/L of biotin was supplemented to the medium. As a result, L: -lactate concentration by the mutant G-03 reached 198.2 g/L (productivity of 5.5 g L(-1) h(-1)) at 41°C in a 7-L fermentor with 210 g/L glucose as carbon source. L: -Lactate concentration and productivity of mutant G-03 were 115.2% and 97.8% higher than those of the parent strain, respectively. The strategy for enhancing L: -lactic acid production by increasing osmotic stress resistance at high temperature may provide an alternative approach to enhance organic acid production with other strains.