Introduction of a cellobiose utilization pathway consisting of a cellodextrin transporter and a β-glucosidase into Saccharomyces cerevisiae enables co-fermentation of cellobiose and xylose. Cellodextrin transporter 1 (CDT1) from Neurospora crassa has been established as an effective transporter for the engineered cellobiose utilization pathways. However, cellodextrin transporter 2 (CDT2) from the same species is a facilitator and has the potential to be more efficient than CDT1 under anaerobic conditions due to its energetic benefits. Currently, CDT2 has a very low activity and is considered rate-limiting in cellobiose fermentation. Here, we report the directed evolution of CDT2 with an increased cellobiose uptake activity, which results in improved cellobiose fermentation under anaerobic conditions. After three rounds of directed evolution, the cellobiose uptake activity of CDT2 was increased by 2.2-fold, which resulted from both increased specific activity and transporter expression level. Using high cell density fermentation under anaerobic conditions, the evolved mutant conferred 4.0- and 4.4-fold increase in the cellobiose consumption rate and ethanol productivity, respectively. In addition, although the cellobiose uptake activity was still lower than that of CDT1, the engineered CDT2 showed significantly improved cellobiose consumption and ethanol production under anaerobic conditions, representing the energetic benefits of a sugar facilitator for anaerobic cellobiose fermentation. This study demonstrated that anaerobic biofuel production could be significantly improved via directed evolution of a sugar transporter protein in yeast. Biotechnol. Bioeng. 2014;111: 1521–1531. © 2014 Wiley Periodicals, Inc.