CLDN4 belongs to a multi-transmembrane protein family of claudins, which mainly functions in cell-cell adhesion and migration. MicroRNAs (miRNAs) are important post-transcriptional regulating factors that participate in broad biological process including immunity. Through high-throughput RNA sequencing strategy, a flounder miRNA, miR-29-x, was identified to be responsible to both bacteria and virus. In this study, we explored the regulatory mechanism and function of miR-29-x and its target gene of flounder CLDN4 (named PoCLDN4). We proved that miR-29-x could interact with the 3'UTR of PoCLDN4 and negatively regulate its expression. PoCLDN4 located on cell membrane, while the depletion of extracellular loop E2 abolished the membrane localization of this protein. E3 could bind different bacteria, and mutation of the amino acids of