Cathepsin F is a unique papain cysteine proteinase with highly conserved structures: catalytic triad and a cystatin domain contained in the elongated N-terminal pro-region. It has been reported that cathepsin F is associated with the establishment of innate immune in several vertebrate including fish in aquaculture, but not known in bivalves. In this study, we firstly identified and characterized cathepsin F in the Yesso scallop (Patinopecten yessoensis). The protein structural and phylogenetic analyses were then conducted to determine its identity and evolutionary position. We've also investigated the expression levels of cathepsin F gene at different embryonic developmental stages, in healthy adult tissues and especially in the hemocytes and hepatopancreas after Gram-positive (Micrococcus luteus) and negative (Vibrio anguillarum) challenges using quantitative real-time PCR (qPCR). Cathepsin F was significantly up-regulated 3 h after infection of V. anguillarum in hemocytes, suggesting its participation in immune response. Our findings have provided strong evidence that cathepsin F may be a good target for enhancing the immune activity in Yesso scallop.