ABSTRACTA highly active xylose specific transporter without glucose inhibition is highly desirable in cost‐effective production of biofuels from lignocellulosic biomass. However, currently available xylose specific transporters suffer from low overall activity and most are inhibited by glucose. In this study, we applied a directed evolution strategy to engineer the xylose specific transporter AN25 from Neurospora crassa with improved xylose transportation capacity. After four rounds of directed evolution using two different strategies, we obtained an AN25 mutant AN25‐R4.18 with 43‐fold improvement in terms of xylose transportation capacity while maintaining its high xylose specificity. In addition, glucose inhibition was almost completely eliminated in the final evolved mutant. We demonstrated that improved xylose transportation of AN25 mutants in the exponential growth phase led to significant improvement of xylose consumption in high cell‐density fermentation. Finally, we showed that AN25 mutant AN25‐R4.18 can enable relatively efficient glucose‐xylose co‐utilization in high concentrations of mixed sugars. Biotechnol. Bioeng. 2016;113: 484–491. © 2015 Wiley Periodicals, Inc.