Ethanol tolerance of self-flocculating yeast SPSC01 was studied in a 3-L bioreactor under fed-batch culture. Yeast floc populations with the average sizes around 100, 200, 300, and 400 μm were obtained by adjusting the mechanical stirring rates of the fermentation system. When subjected to 20% (V/V) ethanol shock for 6 h at 30°C, the remained cell viability was 3.5%、26.7%、48.8% and 37.6% for the aforementioned four floc populations, respectively. The highest ethanol yield 85.5% was achieved for the 300 μm flocs, 7.2% higher than that of the 100 μm flocs. The amounts of trehalose and ergosterol (including free ergosterol and total ergosterol) were positively correlated with the average size distributions from 100 to 300 μm. However, in the 400 μm flocs, the content of trehalose and ergosterol decreased, which coincided with its reduced ethanol tolerance compared to that of the 300 μm flocs. Furthermore, when subjected to 15% (V/V) ethanol shock at 30°C, the equilibrium nucleotide concentration and plasma membrane permeability coefficient(P′) of the 300 μm flocs accounted for only 43% and 52% respectively of those of the 100 μm and 200 μm populations. The effect of floc size distribution on the ethanol tolerance of the self-flocculating yeast strain SPSC01 was closely related to plasma membrane permeability. An optimal floc size distribution with the highest ethanol tolerance and ethanol production level could be obtained by controlling mechanical stirring speed of the bioreactor, which provides basis for the process optimization of fuel ethanol production using this self-flocculating strain.