Aims The aim of this study was to investigate the role of invasin in a bacterial fish pathogen Edwardsiella tarda. Methods and Results In this study, an in-frame deletion mutant of invasin (inv) in Edw.tarda H1 was constructed through double crossover allelic exchange to explore the function of invasin in virulence to fish. Meanwhile, an invasin overexpression strain (inv+) was obtained by electrotransformation of a low-copy plasmid pACYC184 carrying the intact invasin into the inv mutant. Several virulence-associated characters of the mutants and wild-type strain were tested. Compared with the wild-type H1, haemolytic activity and biofilm formation were decreased in inv, while increased significantly in inv+. In addition, the invasin overexpressing strain inv+ exhibited increased internalization into Epithelioma Papulosum Cyprini (EPC) cells. Moreover, in zebrafish model, inv showed decreased virulence compared with H1, while inv+ restored the virulence of wild type completely. Conclusions The results demonstrated that invasin of Edw.tarda plays essential roles in haemolytic activity, biofilm formation, adherence, internalization and pathogenicity of this bacterium. Significance and Impact of the Study This study revealed the role of invasin in Edw.tarda infection and provided useful information for further unveiling the pathogenesis of Edw.tarda.