In this study, we attempted to assess the process stability of long-term fed-batch ethanol fermentation in the absence and presence of aeration (0.33 vvm). To examine the effect of aeration, a long-term repeated fed-batch operation was conducted for 396 h to mimic a long-term industrial bioethanol production process. In this long-term repeated fed-batch ethanol fermentation experiments, withdrawal-fill operation were conducted every 36 h for 10 repeat cycles. The whole operation was stably sustained in a quasi-steady state. The average maximal cell concentration and the average maximal ethanol production during operation were increased by 81.63 and 12.12%, respectively, when aeration was used. In addition, since aeration was carried out, the average ethanol yield slightly decreased by 4.03% and the average specific ethanol production rate decreased by 46.75% during operation. However, the average ethanol productivity increased by 17.53% when aeration was carried out. After 396 h of long-term repeated fed-batch ethanol fermentation, 1,908.9 g of ethanol was cumulatively produced when aeration was used, which was 12.47%, higher than when aeration was not used (1,697.2 g). Meanwhile, glycerol production was greatly decreased during long-term repeated fed-batch ethanol fermentation, in which the glycerol concentration in the culture broth decreased from about 34~15 g/L. Thus, we can conclude that cell growth was greatly improved by overcoming ethanol inhibition and glycerol production was remarkably decreased when aeration was carried out, although aeration in ethanol fermentation decreased the specific ethanol production rate and ethanol yield.