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Whereas the yeast <i>Saccharomyces cerevisiae</i> shows great preference for glucose as a carbon source, a deletion mutant in trehalose-6-phosphate synthase, <i>tps1</i>Δ, is highly sensitive to even a few millimolar glucose, which triggers apoptosis and cell death. Glucose addition to <i>tps1</i>Δ cells causes deregulation of glycolysis with hyperaccumulation of metabolites upstream and depletion downstream of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The apparent metabolic barrier at the level of GAPDH has been difficult to explain. We show that GAPDH isozyme deletion, especially Tdh3, further aggravates glucose sensitivity and metabolic deregulation of <i>tps1</i>Δ cells, but overexpression does not rescue glucose sensitivity. GAPDH has an unusually high pH optimum of 8.0 to 8.5, which is not altered by <i>tps1</i>Δ. Whereas glucose causes short, transient intracellular acidification in wild-type cells, in <i>tps1</i>Δ cells, it causes permanent intracellular acidification. The <i>hxk2</i>Δ and <i>snf1</i>Δ suppressors of <i>tps1</i>Δ restore the transient acidification. These results suggest that GAPDH activity in the <i>tps1</i>Δ mutant may be compromised by the persistently low intracellular pH. Addition of NH<sub>4</sub>Cl together with glucose at high extracellular pH to <i>tps1</i>Δ cells abolishes the pH drop and reduces glucose-6-phosphate (Glu6P) and fructose-1,6-bisphosphate (Fru1,6bisP) hyperaccumulation. It also reduces the glucose uptake rate, but a similar reduction in glucose uptake rate in a <i>tps1</i>Δ <i>hxt2,4,5,6,7</i>Δ strain does not prevent glucose sensitivity and Fru1,6bisP hyperaccumulation. Hence, our results suggest that the glucose-induced intracellular acidification in <i>tps1</i>Δ cells may explain, at least in part, the apparent glycolytic bottleneck at GAPDH but does not appear to fully explain the extreme glucose sensitivity of the <i>tps1</i>Δ mutant.<b>IMPORTANCE</b> Glucose catabolism is the backbone of metabolism in most organisms. In spite of numerous studies and extensive knowledge, major controls on glycolysis and its connections to the other metabolic pathways remain to be discovered. A striking example is provided by the extreme glucose sensitivity of the yeast <i>tps1</i>Δ mutant, which undergoes apoptosis in the presence of just a few millimolar glucose. Previous work has shown that the conspicuous glucose-induced hyperaccumulation of the glycolytic metabolite fructose-1,6-bisphosphate (Fru1,6bisP) in <i>tps1</i>Δ cells triggers apoptosis through activation of the Ras-cAMP-protein kinase A (PKA) signaling pathway. However, the molecular cause of this Fru1,6bisP hyperaccumulation has remained unclear. We now provide evidence that the persistent drop in intracellular pH upon glucose addition to <i>tps1</i>Δ cells likely compromises the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a major glycolytic enzyme downstream of Fru1,6bisP, due to its unusually high pH optimum. Our work highlights the potential importance of intracellular pH fluctuations for control of major metabolic pathways.