The influenza A virus NS1 protein contains a conserved 4-amino-acid-residue PDZ-ligand binding motif (PBM) at the carboxyl terminus that can function as a virulence determinant by targeting cellular PDZ proteins. The NS1 proteins from avian and human viral isolates have consensus PBM sequences ESEV and RSKV, respectively. Currently circulating highly pathogenic H5N1 viruses contain the ESEV PBM which specifically associates with the PDZ proteins Scribble, Dlg1, MAGI-1, MAGI-2, and MAGI-3. In this study, we found NS1 proteins from viral isolates that contain the PBM sequence RSKV, KSEV, or EPEV are unable to associate with these PDZ proteins. Other results showed that the ESEV PBM mediates an indirect association with PDZ protein, Lin7C, via an interaction with Dlg1. Infection with a virus that expresses a NS1 protein with the ESEV PBM results in colocalization of NS1, Scribble, and Dlg1 within perinuclear puncta and mislocalization of plasma membrane-associated Lin7C to the cytoplasm. Infection of polarized MDCK cells with the ESEV virus additionally results in functional disruption of the tight junction (TJ) as measured by altered localization of TJ markers ZO-1 and Occludin, decreased transepithelial electrical resistance, and increased fluorescein isothiocyanate (FITC)-inulin diffusion across the polarized cell monolayer. A similar effect on the TJ was observed in MDCK cells depleted for either Scribble or Dlg1 by small interfering RNA (siRNA). These findings indicate that ESEV PBM-mediated binding of NS1 to Scribble and Dlg1 functions to disrupt the cellular TJ and that this effect likely contributes to the severe disease associated with highly pathogenic H5N1 influenza A viruses.