Collectively, we demonstrate that TLR3 signaling plays a biological role in maintaining epithelial barrier function, and our data implicate Chlamydia infection, TLR3, and the IFN-β synthesized through TLR3 signaling pathways in the assembly and stability of cellular tight junctions during Chlamydia infection of OE cell monolayers. Here, TLR3 is linked to chlamydia infectious disease.