Evolution of bacterial tolerance to antimicrobials precedes evolution of resistance and may result in cross-tolerance, cross-resistance or collateral sensitivity to other antibiotics. Transient exposure of gut bacteria to glyphosate, the world's most widely used herbicide, has been linked to the activation of the stress response and changes in susceptibility to antibiotics. In this study, we investigated whether a chronic exposure to a glyphosate-containing herbicide (GBH) results in resistance, a constitutive activation of the tolerance and stress response and cross-tolerance or cross-resistance to antibiotics. Of the ten farm animals-derived clinical isolates of Salmonella enterica subjected to experimental evolution in increasing concentrations of GBH, three isolates showed a stable resistance with mutations associated with the glyphosate target gene aroA and no fitness costs. The global quantitative proteomics analysis demonstrated activation of the cellular tolerance and stress response during the transient exposure to GBH, but not constitutively in the resistant mutants. Resistant mutants displayed no cross-resistance or cross-tolerance to antibiotics. These results suggest that while transient exposure to GBH triggers cellular tolerance response in Salmonella enterica, this response does not become genetically fixed after selection for resistance to GBH and does not result in increased cross-tolerance or cross-resistance to clinically important antibiotics under our experimental conditions. Importance Glyphosate-based herbicides (GBH) are among the world's most popular, with traces commonly found in food, feed and the environment. Such high ubiquity means that the herbicide may come into contact with various microorganisms, on which it acts as an antimicrobial and may select for resistance and cross-resistance to clinically important antibiotics. It is therefore important to estimate whether the widespread use of pesticides may be an underappreciated source of antibiotic resistant microorganisms that may compromise efficiency of antibiotic treatments in humans and animals.