Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters and, in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose (HXT) transporters. By means of in vivo engineering, we have evolved a quadruple hexokinase deletion mutant of S. cerevisiae into a strain that efficiently utilizes D-xylose in the presence of high D-glucose concentrations. Genome sequence analysis revealed a mutation (Y353C) in the general co-repressor CYC8/SSN6, which was found to be responsible for the phenotype when introduced individually in the non-evolved strain. Transcriptome analysis revealed the altered expression of in total 95 genes, including genes involved in: 1) hexose transport, 2) maltose metabolism, 3) cell wall function (mannoprotein family), and 4) unknown functions (Seripauperin multigene family). Out of the 18 known HXT transporters, 9 were upregulated, especially the low or non-expressed HXT10, HXT13, HXT15 and HXT16 genes. Mutant cells show increased uptake rates of D-xylose in the presence of D-glucose, as well as an elevated Vmax for both D-glucose and D-xylose transport. The data suggests that the increased expression of multiple hexose transporters renders D-xylose metabolism less sensitive to D-glucose inhibition due an elevated transport rate of D-xylose into the cell.IMPORTANCE The yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous Hxt transporter as uptake is outcompeted by the preferred substrate glucose. Mutants were obtained with improved growth characteristics on xylose in the presence of glucose, and map to the regulator Cyc8. The inactivation of Cyc8 cause the increased expression of Hxt transporters thereby providing more capacity for the transport of xylose as well, presenting a further step towards a more robust process of industrial fermentation of lignocellulosic biomass using yeast.