Protease inhibitors are widely distributed in the skin mucus of fish. The pufferfish, family Tetraodontidae, has high antitryptic activity among fish. In this study, we isolated and characterized a protease inhibitor from the skin mucus of the pufferfish Takifugu rubripes. The inhibitor termed T. rubripes trypsin inhibitor 2 (TRTI-2) is a serine protease inhibitor and specifically inhibits trypsin with the inhibitory constant (Ki) of 8.6 × 10–8 M. TRTI-2 is an acidic glycoprotein with a molecular mass of 63 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and deglycosylated to 46 kDa by treatment with glycopeptidase. Amino acid sequence and cDNA cloning analyses revealed that mature TRTI-2 comprises 421 amino acid residues with a theoretical molecular mass of 47,349.17 Da and isoelectric point (pI) of 5.47. A BLAST homology search revealed that TRTI-2 is homologous to hemopexin family protein and identical with fWap65-2, the warm temperature acclimation-related 65-kDa protein (Wap65) from T. rubripes. Wap65 shows functional diversification as a mediator of not only temperature acclimation but also inflammatory, antiinflammatory, and immune responses. The present study revealed a novel biochemical property of Wap65 as an antitrypsin, possibly to protect the functional molecule from damage by protease.