At present, very little is known about the development of tolerance in response to osmotic stress. Although cells undergo similar adaptations to both antibiotic and osmotic stress, there exists a need to elucidate the relationship, observed through growth rate patterns, metabolic perturbations, and membrane alterations, in response to simultaneous exposure to both stresses. A better understanding of these interactions will produce new strategies for treating persistent and recalcitrant bacterial infections. We show here that osmotic stress cross-protects E. coli to bactericidal antibiotics, conferring tolerance to antibiotics.