Porphyromonas gingivalis is a major pathogen in severe and chronic manifestations of periodontal disease, which is one of the most common infections of humans. A central feature of P. gingivalis pathogenicity is dysregulation of innate immunity at the gingival epithelial interface. We previously showed that junctional adhesion molecule 1 (JAM1) was specifically degraded by P. gingivalis, leading to epithelial barrier breakdown in gingival tissues. Whereas, the involvement of the other JAM family protein(s) in the epithelial barrier dysregulation by P. gingivalis remains unknown. Here we show that Arg-specific or Lys-specific cysteine proteases named gingipains produced by P. gingivalis specifically degrade coxsackievirus and adenovirus receptor (CXADR), a tight junction associated protein, at R145 and K235 in gingival epithelial cells. A P. gingivalis strain lacking gingipains was impaired in degradation of CXADR. Knockdown of CXADR in a three-dimensional multilayered tissue model increased permeability to 40 kDa dextran, lipopolysaccharide, and proteoglycan. Inversely, overexpression of CXADR in a gingival epithelial tissue model prevented penetration by these agents following P. gingivalis infection. Our findings strongly suggest that P. gingivalis gingipains disrupt barrier function of stratified squamous epithelium via degradation of CXADR as well as JAM1, efficiently allowing bacterial virulence factors to penetrate into subepithelial tissues.