Kunitz-type serine protease inhibitor (KSPI) interacts with serine protease (SP) to regulate cascade reactions in vivo and plays essential roles in innate immunity. Theoretical considerations support various functions of kspi, but further studies are required for full characterization of these functions. In this study, a KSPI molecule was identified from Japanese flounder (Paralichthys olivaceus), and was named Pokspi. The full-length cDNA sequence of Pokspi was 2810 nt, containing an open reading frame of 1527 nt, which encoded a polypeptide of 509 amino acid residues. PoKspi protein contained five conversed domains, namely, MANEC, PKD, LDLa and two Kunitz domains. Homology analysis revealed that Pokspi shared the highest similarity (83%) with its homolog in Cynoglossus semilaevis. Phylogenetic analysis indicated that Pokspi clustered with the homologs in other fishes. The mRNA transcripts of Pokspi were detected in all tested tissues, with the highest expression level in gill, followed by kidney and intestine. Its elevated expression in response to the application of Edwardsiella tarda (in vivo) and pathogen-associated molecular pattern (in vitro) suggested the involvement of Pokspi in the essential immune defense against various pathogens. Recombinant PoKspi (rPoKspi) purified from Escherichia coli exhibited not only serine protease inhibitor activities but also a broad spectrum of anti-microbial effect in a manner that was independent of any host factors. In addition, the recombinant PoKspi protein could cause the down-regulation of pro-inflammatory factors TNF-α and IL-1β. In conclusion, Pokspi is a biologically active serine protease inhibitor endowed with anti-bacterial and anti-inflammatory property. This study provides strong evidences for understanding the innate immune defense in Japanese flounder.