To further increase the rate of glucose consumption by multi-vitamin auxotrophic yeast Torulopsis glabrata. A neomycin-resistant mutant N07, with the activity of F1-ATPase decreased roughly 35% but glucose consumed per cell was increased 38% than that of parent strain, was breed based on analysis of energy metabolic pathway. The typical inhibitors of F1F0-ATPase, DCCD, NaN3 and neomycin, depressed the F1-ATPase activity of parental strain but no effect on that of mutant strain. Strain N07 was cultured in a pyruvate fermentation medium containing 100g/L of glucose using flask. It was found that the rate of glucose consumption and pyruvate production were higher by 34% and 42.9% in the mutant than in the parent, respectively. However, the rate and yield of growth (about 24%) of the mutant was lower than that of the parent. The content of intracellular ATP of the mutant also decreased 23.7% than that of the parent. The activities of key enzymes in glycolytic pathway and electron transfer chain of the mutant and the parent were determined. Enzymatic analysis revealed that, compared with the parent strain CCTCC M202019. The activities of key enzymes, phosphofructokinase, pyruvate kinase, glyceraldyde-3-phosphate dehydrogenase of the mutant N07 increased 63.7%, 28.8% and 14.4%, respectively, all the key enzymes of electron transfer chain in the mutant N07 also increased roughly 10%.