Tight junctions (TJ) are structurally defined by networks of linear sealing strands between adjoining epithelial cells; this structure regulates the ion and solute permeation through the intercellular space. Claudins, a large family of transmembrane proteins, are the main components of the TJ strand network. A crystal structure of mclaudin-15 monomer was reported recently; however, the crucial domains facilitating polymerization into highly ordered, yet flexible, strands still remain unknown. In this study, we aimed to identify the crucial claudin protomer interfaces required for polymerization using both in silico and in vitro methods. First, we use computational methods including evolutionary couplings, conservation analysis, and solvent accessible surface area evaluation to identify potential key residues on the oligomerization interface. We then perform mutagenesis analysis, confocal imaging of TJ formation in transfected cultured cells, and freeze fracture EM to test the in silico data. We further use molecular docking to construct claudin oligomeric models and molecular dynamics to study intermolecular interactions at the atomic level. Two distinct intermolecular interfaces were obtained after an extensive screening of 1200 interfaces during in silico docking using in silico and in vitro data as constraints. Specifically, hydrophobic residues in extracellular loop1 and loop2 form a “head-to-head” (-trans) interface. In addition, intermolecular hydrogen bonds between hydrophilic residues on the extracellular helix and charged residues on the β-strand (β5) form an axial (-cis) interface. Mutations of these residues eliminated, reduced, or weakened the TJ strands. Combining the data obtained from these different approaches, we further propose an octameric extracellular pore model, which is consistent with the selectivity, size (∼10 A), and evidence for pore-lining residues reported previously. Our study provides mechanistic insight into TJ polymerization, paracellular molecular flow through TJ, and potential targets for TJ modulation.