This study investigates the immunomodulatory effects of phenoloxidase (PO), purified from the hemolymph of Haliotis discus hannai, a commercially valuable seafood consumed raw in Asia. We aimed to elucidate the impact of PO on human colonic epithelial cells (HT-29) exposed to lipopolysaccharide (LPS), a pro-inflammatory component of Gram-negative bacteria. PO was successfully purified from H. discus hannai hemolymph and exhibited no cytotoxicity towards HT-29 cells at concentrations up to 200 μg/mL. Treatment with PO significantly reduced free LPS levels, indicating its potential role in mitigating bacterial challenge. Furthermore, PO treatment alongside LPS stimulation in HT-29 cells effectively regulated the production of pro-inflammatory cytokines, cytokines associated with hyperimmune responses, and prostaglandin 2 (PGE2), suggesting its ability to modulate inflammatory responses. Mechanistic investigation suggests that PO might exert its anti-inflammatory effects by regulating the ERK1/2-mediated NF-κB pathway. These findings highlight the potential anti-inflammatory properties of H. discus hannai hemolymph and its PO component, thus Further in depth studies on the specific structure of PO and its interaction with inflammation inducers such as LPS are needed.