The probiotic Lactobacillus plantarum LIP-1 was cultured in modified MRS medium at high cell density to investigate the effect of changes in medium components (the types and amounts of nitrogen source, carbon source and buffer salt, total amount of carbon and nitrogen, and carbon/nitrogen ratio) on the viable cell count and survival rate after freezedrying of this strain. The optimal medium was found to be composed of carbon source (sucrose) 33.35 g/L, nitrogen source (yeast powder, soy peptone and casein peptone, 3:3:5, m/m) 10.80 g/L, and buffer salt (sodium acetate, potassium dihydrogen phosphate and sodium citrate, 2.5:1:1, m/m) 14.85 g/L (namely 0.13 mol/L). The viable cell count of strain LIP-1 cultured in the optimized medium after freeze-drying was 9.767 (lg(CFU/mL)), which was distinctly higher than that cultured in MRS medium. The modified medium allowed high-cell-density fermentation of Lactobacillus plantarum LIP-1 while maintaining its viability during freeze-drying.