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The aim of the present study was to elucidate the effects of the probiotic feed additive Enterococcus faecium NCIMB 10415 (E. faecium) on porcine jejunal epithelial cells (IPEC-J2) during an in vitro challenge with enterotoxigenic Escherichia coli (ETEC). Cells were incubated with E. faecium, ETEC, or both, and the effects on barrier function and structure and intra- and intercellular signaling were determined. Coincubation with E. faecium abolished the ETEC-induced decrease in transepithelial resistance (Rt) (p ≤ 0.05). No differences were seen in the expression levels of the intercellular connecting tight junction proteins examined. However, for the first time, a reorganization of the monolayer was observed in ETEC-infected cells but not in coincubated cells. ETEC induced an increase in cytotoxicity that was prevented by coincubation (p ≤ 0.05), whereas apoptosis rates were not affected by bacterial treatment. ETEC increased the mRNA expression and release of proinflammatory cytokines TNF-α, IL-1α, and IL-6 which could be prevented by coincubation for TNF-α mRNA expression and IL-6 protein (p ≤ 0.05). Likewise, cAMP concentrations elevated by ETEC were reduced in coincubated cells (p ≤ 0.05). These findings indicate a protective effect of the probiotic E. faecium on inflammatory responses during infection with ETEC.