Background: A dysfunctional intestinal epithelial tight junction (TJ) barrier is a pathogenic factor in inflammatory bowel disease, and enhancement or protection is an important therapeutic target. Lactobacillus acidophilus (LA) is a common probiotic bacterial strain that has been shown to induce anti-inflammatory effects in the gastrointestinal tract. Our lab recently showed that LA caused an enhancement of the intestinal TJ barrier mediated by attachment of live LA to toll-like receptor-2 (TLR2) at the apical membrane of enterocytes but the adaptor proteins involved remains unclear. Aim: The aim of this study was to delineate the TLR2 signaling mechanisms mediating the LA-induced enhancement of the intestinal TJ barrier. Methods: An in vitro model system consisting of filter-grown Caco-2 monolayers and an in vivo model system of recycling intestinal perfusion of live mice were used to assess intestinal TJ permeability. Results: 1) LA treatment (1×108 cfu/ml) caused an enhancement of the intestinal epithelial TJ barrier as measured by an increase in Caco-2 transepithelial resistance (TER) that occurred in a strain-specific manner. 2) The LA-induced increase in TER in Caco-2 monolayers was mediated by LA binding to TLR2 at the apical cell membrane with an associated increase in the protein expression of the TLR2 adaptor protein TIR Domain Containing Adaptor Protein (TIRAP). 3) siRNA knockdown of TIRAP prevented the LA-induced enhancement of Caco-2 intestinal epithelial TJ barrier. LA did not cause an increase in MyD88 expression or activity (IRAK4 phosphorylation), and siRNA induced knock-down of MyD88 did not prevent the LA enhancement of intestinal epithelial TJ barrier in Caco-2 monolayers. 4) LA caused enhancement of mouse intestinal permeability, but the effect was prevented in TLR2 deficient mice, as assessed by dextran 10K flux in live mice. 5) Although MyD88 is a key adaptor protein in mediating the LA-induced TLR2 signaling pathway in immune cells ex vivo, LA-induced increase in intestinal epithelial TJ barrier function was not prevented in MyD88 deficient mice. Conclusions: Our data showed that LA causes a strain-specific enhancement of the intestinal epithelial TJ barrier by a TLR2-dependent mechanism. In contrast to TLR2-mediated signaling in immune cells, the LA/TLR2 enhancement of intestinal TJ barrier was independent of MyD88, but dependent of TIRAP, suggesting a novel mechanism of intestinal TJ barrier regulation.