The gut forms a vital niche for the survival and replication of drug-resistant E. coli; however, the role of gut conditions on drug-resistant and sensitive E. coli is not clearly understood. The study aims to understand the effect of in vitro gut conditions on the spread of antibiotic resistance among E. coli and their ability to adapt to gut conditions. In this study, a multidrug-resistant (J51) and a sensitive (J254) E. coli isolate were exposed to a series of in vitro gut conditions and their growth pattern, virulence gene expression and invasion ability were studied. Further, the effect of antibiotic under in vitro gut conditions was also studied. Bile significantly affected the growth of the isolates, and the addition of iron chelator extended the lag phase of the sensitive isolate. Each in vitro gut condition had a differential effect on the expression of virulence genes in both the isolates. Further, the resistant isolate could adhere to and invade Caco2 cell lines better than the sensitive isolate. Most of the downregulated genes showed increased expression upon ciprofloxacin shock under in vitro gut conditions. The transcriptomics study revealed that exposure to bile, led to the downregulation of genes involved in different metabolic pathways. Further downregulation of metabolic pathways on ciprofloxacin shock was also observed. The downregulation of metabolic pathways could be a part of the global response played by the bacteria to adapt to harsh conditions. Reverting these fluctuated pathways could prove to be a novel strategy in combating AMR threat. Overall, bile, in high and low temperature conditions, showed a significant effect on modulating virulence gene expression on the antibiotic challenge. Thus, it is essential to consider the impact of gut conditions on gut pathogens, such as E. coli, before prescribing antimicrobial therapy during infection.