Antibiotics have been widely included in feeds to prevent post-weaning diarrhea (PWD) and increase the overall growth performance of pigs. However, there is a trend to minimize or eliminate the use of in-feed antibiotics. The development of effective alternatives to in-feed antibiotics (e.g., probiotics) is crucial for maintaining the sustainability of swine production. For young piglets, an effective probiotic is expected to deliver at least one of the following functions to the gut: 1) stimulating the development of a healthy microbiota - predominated by beneficial bacteria, 2) preventing enteric pathogens from colonization, 3) increasing digestive capacity and lowering the pH, 4) improving mucosal immunity, or 5) enhancing gut tissue maturation and integrity. Our previously isolated lactate-producing bacteria (e.g. Lactobacillus zeae LB1) have shown to prevent Caenorhabditis elegans (C. elegans) from enterotoxigenic Escherichia coli F4 (ETEC F4)-related death by inhibiting enterotoxin gene expression of the pathogen rather than interfering with its intestinal colonization. However, the protective effects of L. zeae LB1 at cellular and molecular levels have not been investigated yet in pigs. In the present study, porcine intestinal cells (IPEC-J2) were used to investigate the potential of L. zeae LB1 on modulating intestinal barrier and innate immune functions and protecting against intestinal injuries and inflammatory reactions induced by ETEC F4 infection. The results suggested that probiotic L. zeae LB1 effectively protected the intestinal cells from ETEC F4 infection by inhibiting inflammation and maintaining barrier integrity via downregulating TLR4, TLR5, and ETEC F4 virulence-related factors expressions. In conclusion, our data provide further evidence on the mechanisms at the cellular and molecular levels that probiotic L. zeae LB1 may improve gut health by enhancing barrier function and reducing inflammatory cytokines secretion in pigs under physiological challenges.