Antibiotic tolerance, the widespread ability of diverse pathogenic bacteria to sustain viability in the presence of typically bactericidal antibiotics for extended time periods, is an understudied stepping stone toward antibiotic resistance. The gram-negative pathogen Vibrio cholerae, the causative agent of cholera, is highly tolerant to β-lactam antibiotics. We previously found that the disruption of glycolysis, via deletion of pgi (vc0374, glucose-6-phosphate isomerase), resulted in significant cell wall damage and increased sensitivity toward β-lactam antibiotics. Here, we uncover the mechanism of this resulting damage. We find that glucose causes growth inhibition, partial lysis, and a damaged cell envelope in ∆pgi. Supplementation with N-acetylglucosamine, but not other carbon sources (either from upper glycolysis, TCA cycle intermediates, or cell wall precursors) restored growth, re-established wild-type β-lactam resistance, and recovered cellular morphology of a pgi mutant exposed to glucose. Targeted metabolomics revealed the cell wall precursor synthetase enzyme GlmU (vc2762, coding for the bifunctional enzyme that converts glucosamine-1P to UDP-GlcNAc) as a critical bottleneck and mediator of glucose toxicity in ∆pgi. In vitro assays of GlmU revealed that sugar phosphates (primarily glucose-1-phosphate) inhibit the acetyltransferase activity of GlmU (likely competitively), resulting in compromised peptidoglycan and lipopolysaccharide biosynthesis. These findings identify the molecular mechanism of Δpgi glucose toxicity in V. cholerae.