To verify the roles of GltS, GltP, and GltI in E. coli tolerance and pathogenicity, we quantified and compared the relative abundance of gltS, gltP, and gltI in log-phase and stationary-phase E. coli and constructed their knockout mutant strains in E. coli BW25113 and uropathogenic E. coli (UPEC) separately, followed by analysis of their abilities to tolerate antibiotics and stressors, their capacity for adhesion to and invasion of human bladder epithelial cells, and their survival ability in mouse urinary tracts. Our results showed that gltS, gltP, and gltI transcripts were higher in stationary phase E. coli than in log-phase incubation. Furthermore, deletion of gltS, gltP, and gltI genes in E. coli BW25113 results in decreased tolerance to antibiotics (levofloxacin and ofloxacin) and stressors (acid pH, hyperosmosis, and heat), and loss of gltS, gltP, and gltI in uropathogenic E. coli UTI89 caused attenuated adhesion and invasion in human bladder epithelial cells and markedly reduced survival in mice. The results showed the important roles of the glutamate transporter genes gltI, gltP, and gltS in E. coli tolerance to antibiotics (levofloxacin and ofloxacin) and stressors (acid pH, hyperosmosis, and heat) in vitro and in pathogenicity in mouse urinary tracts and human bladder epithelial cells, as shown by reduced survival and colonization, which improves our understanding of the molecular mechanisms of bacterial tolerance and pathogenicity.