This study was aimed at increasing the glycolytic flux of the multivitamin-auxotrophic yeast Torulopsis glabrata by disturbing oxidative phosphorylation. We examined two different strategies to impede oxidative phosphorylation. The first strategy was disruption of the activity of the electron transfer chain (ETC), by either of two approaches. One was separately adding, at 10 mg L1, specific inhibitors of complex I (rotenone) or of the bc1 complex (antimycin A) to the culture broth of T. glabrata CCTCC M202019, which resulted in significantly decreased intracellular ATP levels (43% and 27.7%) and significantly increased rates of glucose consumption (qs) and pyruvate production (qp); another approach was breeding a respiratory-deficient mutant RD-16, in which cytochromes aa3 and b in the ETC were deleted after ethidium bromide mutagenesis, to reduce the ETC activity constitutively. The second strategy was inhibiting F0F1-ATP synthase with 0.05 mM oligomycin. Also, a neomycin-resistant mutant with 65% decreased F0F1-ATPase activity was studied. With the two strategies, the specific activity of phosphofructokinase (R2=0.9971), the average specific glucose consumption rate (R2=0.9967) and the average specific pyruvate production rate (R2=0.965) were closely correlated with the intracellular ATP level, all of them being increased at a lower intracellular ATP level.