Porins are crucial proteins located in the outer membrane that directly influence antimicrobial resistance mechanisms and virulence in bacteria. In this study, a porin gene (Vp-porin) was cloned in V. parahaemolyticus, and the function of Vp-porin on biological characteristics and virulence was investigated. The results of sequence analysis showed that Vp-porin is highly conserved in Vibrio spp. and the predicted 3D structure showed it could form a transmembrane channel with 20 β barrel. Membrane permeabilization provides evidence that membrane integrity of ∆Vp-porin was damaged and the sensitivity to tetracycline, polymyxin B, rifampicin and cephalothin of ∆Vp-porin obvious increased. In addition, loss of Vp-porin damaged motility due to down-regulated flagellar synthesis. Besides, ∆Vp-porin exhibited attenuated cytotoxicity to Tetra-hymena. The relative survival rate of Tetrahymena infection with ∆Vp-porin was 86%, which is much high than that with WT (49%). Take together, Vp-porin in V. parahaemolyticus plays various roles in biological characteristics in membrane integrity, antimicrobial resistance and motility, and contributes to virulence.