In order to economically produce biofuels in simultaneous saccharification and fermentation (SSF) of cellulosic biomass, it is required to construct microbial host efficiently utilizing various sugars. Previously, a mutant Pichia stipitis YN14 strain co-fermenting cellobiose and xylose was isolated. In this study, ethanol production performance of P. stipitis YN14 was compared with the parental P. stipitis CBS 6054 through the series of SSF experiments. In SSF of cellulose, the mutant strain employing Celluclast 1.5 L as the cellulolytic enzyme produced 6.1 g/L of ethanol, which was 5.5- times and 1.2-times higher than the parental strain with Celluclast 1.5 L and Cellic CTec2, respectively. In addition, the mutant YN14 strain with Celluclast 1.5 L showed 26.6 g/L of ethanol production in simultaneous saccharification and cofermentation (SSCF) of cellulose and xylose, which was 75% and 60% higher than the parental CBS 6054 strain with Celluclast 1.5 L and Cellic CTec2, respectively. Our results propose that efficient ethanol production from the cellulosic biomass would be accomplished by utilization of yeast strain fermenting cellobiose and xylose simultaneously.