To verify the roles of GltS, GltP, and GltI inE. colitolerance and pathogenicity, we quantified and compared the relative abundance ofgltS,gltP, andgltIin log-phase and stationary-phaseE. coliand constructed their knockout mutant strains inE. coliBW25113 and uropathogenicE. 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 thatgltS,gltP, andgltItranscripts were higher in stationary phaseE. colithan in log-phase incubation. Furthermore, deletion ofgltS,gltP, andgltIgenes inE. coliBW25113 results in decreased tolerance to antibiotics (levofloxacin and ofloxacin) and stressors (acid pH, hyperosmosis, and heat), and loss ofgltS,gltP, andgltIin uropathogenicE. coliUTI89 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 genesgltI,gltP, andgltSinE. colitolerance 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.