Pinctada fucata martensii is one of the principal shellfish species used for marine pearl cultivation in China and holds considerable economic value. However, increasing pollution in mariculture environments has heightened its susceptibility to pathogenic bacterial infections, causing large-scale mortality that severely limits sustainable aquaculture development. Previous work identified PmFREP1, a fibrinogen-related protein highly expressed in gill tissue that responds markedly to immune stimulation, suggesting a role in early pathogen recognition. To characterize its antibacterial and agglutination at the protein level, we produced recombinant PmFREP1 using a prokaryotic expression system and further generated a polyclonal antibody targeting this protein. In vitro assays demonstrated that rPmFREP1 significantly inhibited growth of multiple bacteria, with the strongest effects against Vibrio parahaemolyticus (49% inhibition rate) and Staphylococcus aureus (69% inhibition rate), and mediated Ca2+-dependent bacterial agglutination. Polyclonal antibodies against rPmFREP1 detected PmFREP1-positive signals in gill tissue by immunohistochemistry. In vivo, co-injection of rPmFREP1 with V. parahaemolyticus preserved 88.33% survival at day 10, while anti-rPmFREP1 antibody blockade led to 100% mortality by day 21. Collectively, these findings suggest that PmFREP1 participates in antibacterial defense in P. f. martensii by contributing to bacterial growth inhibition and Ca2+-enhanced agglutination, providing experimental evidence for FREP-mediated innate immune defense in bivalves.