Glycolysis and the pentose phosphate pathway (PPP) are two basic metabolic pathways that are simultaneously present in yeasts. As the main pathway in most species, the glycolysis provides ATP and NADH for cell metabolism while PPP, as a complementary pathway, supplies NADPH. In this study, the performance ofKluyveromyces marxianususing glycolysis or PPP were studied through the disruption ofPGI1orZWF1gene, respectively.K. marxianususing glycolysis as the only pathway showed higher ethanol production ability than that of theKluyveromyces lactis zwf1 Delta mutant;K. marxianususing only PPP showed more robustness than that ofSaccharomyces cerevisiae pgi1 Delta mutant. Additionally,K. marxianus pgi1 Delta strain accumulated much more intracellular NADPH than the wild type strain and co-utilized glucose and xylose more effectively. These findings suggest that phosphoglucose isomerase participates in the regulation of the repression of glucose on xylose utilization inK. marxianus. The NADPH/NADP(+)ratio, dependent on the activity of the PPP, regulated the expression of multiple genes related to NADPH metabolism inK. marxianus(includingNDE1,NDE2,GLR1, andGDP1). SinceK. marxianusis considered a promising host in industrial biotechnology to produce renewable chemicals from plant biomass feedstocks, our research showed the potential of the thermotolerantK. marxianusto produce NADP(H)-dependent chemical synthesis from multiple feedstocks.