Ethanol tolerance is a key trait of microbes in bioethanol production. Previous studies have shown that soya flour contributed to the increase of ethanol tolerance of yeast cells. In this paper, the mechanism of this ethanol tolerance improvement was investigated in cassava ethanol fermentation supplemented with soya flour or defatted soya flour, respectively. Experiment results showed that ethanol tolerance of cells from soya flour supplemented medium increased by 4-6% (v/v) than the control with defatted soya flour. Microscopic observation found that soya flour can retain the cell shape while dramatic elongations of cells were observed with the defatted soya flour supplemented medium. Unsaturated fatty acids (UFAs) compositions of cell membrane were analyzed and the UFAs amounts increased significantly in all tested strains grown in soya flour supplemented medium. Growth study also showed that soya flour stimulated the cell growth rate by approximately tenfolds at 72-h fermentation. All these results suggested that soya fatty acids play an important role to protect yeast cells from ethanol stress during fermentation process.