It is one serious problem that high sugar concentration at an initial phase and/or accumulation of product during cultivation causes osmotic stress to cells in fermentation process using yeast, Saccharomyces cerevisiae. In this study, we analyzed the protein expression of yeast using two-dimensional electrophoresis under a high osmotic pressure condition and investigated the physiological change in yeast cell. A laboratory strain FY834 and brewing one IFO2347 were grown in YPD medium. High osmotic pressure condition was prepared by adding NaCl at final concentration of 1 M in mid-log phase. Although the growth of FY834 was stopped and that of IFO2347 was decreased, the growth of both strains was recovered later. The period between addition of NaCl and recovery of growth was 4 hours for FY834 and 2 hours for IFO2347, respectively, indicating that IFO2347 was more tolerant to osmotic stress than FY834. Protein samples were extracted from cells before and after adding NaCl and applied to two-dimensional electrophoresis. Protein expression of FY834 compared to the sample before addition of NaCl was changed after 30 min from adding NaCl, while that of IFO2347 was changed after 15 min. These results suggest that IFO2347 responds more quickly to osmotic pressure than FY834. From the view point of the metabolic pathway, the expression of proteins related to glycerol synthesis were increased after addition of NaCl in both strains, suggesting that yeast strains adapt to high osmotic pressure by synthesis of glycerol. It was consistent to the results of metabolic flux analysis.