One way of overcoming the substrate and ethanol inhibition effects in the industrial ethanol production process is to use fed-batch fermentation coupled with an ethanol removal technique. This work describes the optimization and experimental validation of sugar cane ethanol production by fed-batch fermentation with in situ ethanol removal by CO, stripping. The optimization employing a genetic algorithm (GA) was used to find the optimum feed flow rate (F) and the ethanol concentration (C-E0) in the medium at which to initiate stripping, in order to obtain maximum ethanol productivity. Conventional ethanol fermentation employing the optimum feed flow rate was performed with must containing 257.1 g L-1 of sucrose (180 g L-1 of total sucrose concentration), resulting in achievement of an ethanol concentration of 82.2 g L-1. The stripping fed-batch fermentation with high total sucrose concentration (260-300 g L-1) or 371.4-428.6 g L-1 in the must feeding was performed with optimal values of the feed flow rate and the ethanol concentration (C-E0) in the medium at which to initiate stripping. At the highest sucrose feed (total concentration of 300 g L-1), the total ethanol concentration reached 136.9 g L-1 (17.2 degrees GL), which was about 65% higher than the value obtained in fed-batch fermentation without ethanol removal by CO, stripping. This strategy proved to be a promising way to minimize inhibition by both the substrate and ethanol, leading to increased sugar cane ethanol production, reduced vinasse generation, and lower process costs.