Vitronectin (Vtn) is a multifunctional protein that plays significant roles in cell adhesion, migration, spreading and survival, and in the regulation of membrane attack complex formation and the terminal pathway of complement activation in innate immune response. However, the expression and immune significance of Vtn in fish remains largely unknown. In order to understand the involvement of Vtn in fish innate immune response, here we cloned and characterized a full-length Vtn ortholog cDNA, termed PoVtn, from Japanese flounder Paralichthys olivaceus. The deduced PoVtn protein is comprised of 438 amino acids with a 19-amino-acid signal peptide sequence (1Met-19Ala) at the N-terminus. Protein domain analysis revealed that PoVtn possesses a conserved N-terminal somatomedin B domain followed by a conserved RGD motif and four haemopexin-like domains. Sequence analysis revealed that PoVtn has two potential glycosylation sites and shares 44–74% sequence identity with other teleost Vtn proteins. PoVtn mRNA was ubiquitously distributed in all examined normal tissues and showed the highest expression in Japanese flounder hepatopancreas tissue. PoVtn expression was induced by LPS and poly(I:C) challenges in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood leucocytes (PBLs) and shows a pathogen-associated molecular pattern- and cell type-dependent manner. The expression of PoVtn was also modulated by bacterial challenge with Edwardsiella tarda in Japanese flounder immune-related tissues including head kidney, gill and spleen. Furthermore, overexpression of PoVtn in Japanese flounder FG-9307 cells significantly attenuated the LPS- and poly(I:C)-induced proinflammatory cytokines IL-1beta and TNF-alpha gene expression. Taken our findings together, we for the first time characterized Vtn gene expression in response to inflammatory stimuli in fish. Our results suggested a potential role of PoVtn in regulating fish innate immunity.