Probiotics are microbes having tremendous potential to prevent gastrointestinal disorders. In current investigation, immunomodulatory action of probiotic Lacticaseibacillus rhamnosus MTCC-5897 was studied during exclusion, competition and displacement of Escherichia coli on intestinal epithelial (Caco-2) cells. The incubation of intestinal cells with Escherichia coli, enhanced downstream signalling and activated nuclear factor kappa B (NF-κB). This significantly increased (p < 0.01) the pro-inflammatory cytokines (IL-8, TNF-α, IFN-ϒ) expression. While, incubation of epithelial cells with Lacticaseibacillus rhamnosus during exclusion and competition with Escherichia coli, counteracted these enhanced expressions. The immunomodulatory feature of Lacticaseibacillus rhamnosus was also highlighted with increased (p < 0.05) transcription of toll-like receptor-2 (TLR-2) and single Ig IL-1-related receptor (SIGIRR) along with diminished expression of TLR-4. Likewise, attenuation (p < 0.05) of E. coli-mediated enhanced nuclear translocation of NF-κB p-65 subunit by Lacticaseibacillus rhamnosus during exclusion was confirmed with western blotting. Thus, present finding establishes the prophylactic potential of Lacticaseibacillus rhamnosus against exclusion of Escherichia coli in intestinal cells.