Abstract: Free fatty acid receptor 4 (FFAR4), a G protein-coupled receptor (GPCR), plays a key role in sensing long-chain polyunsaturated fatty acids (LC-PUFAs) in mammals. However, its ligand selectivity and downstream signaling mechanisms in fish remain poorly understood. In this study, we investigated the ligand recognition and signaling characteristics of FFAR4 from the large yellow croaker (Larimichthys crocea), with particular emphasis on ω-3 and ω-6 fatty acids. A stable HEK293T cell line expressing LcFFAR4 was established to systematically assess ligand-induced receptor internalization, intracellular calcium flux, and cyclic adenosine monophosphate (cAMP) signaling pathway. Subcellular localization analysis revealed predominant plasma membrane distribution of LcFFAR4. Ligand stimulation assays demonstrated that both eicosapentaenoic acid (EPA, ω-3) and linoleic acid (LA, ω-6) induced receptor internalization, with EPA showing significantly stronger effect. Calcium imaging revealed low concentrations EPA markedly increased intracellular calcium levels, indicating activation of the calcium signaling pathway, whereas LA elicited no significant change. Dual-luciferase reporter assays further confirmed both EPA and LA activated the LcFFAR4-mediated cAMP signaling pathway, with EPA displaying a more potent effect. Functional analyses revealed that EPA more effectively mitigated lipopolysaccharide (LPS)-induced inflammatory responses via LcFFAR4. These results collectively demonstrate that LcFFAR4 exhibits distinct ligand selectivity and signaling bias. EPA, an ω-3 fatty acid, functions as a more potent agonist than LA, inducing stronger receptor internalization and more robust activation of both calcium and cAMP signaling pathways. This study provides the first evidence of teleost FFAR4 differential responses to ω-3 and ω-6 fatty acids from a GPCR signaling perspective, offering new insights into the fatty acid sensing mechanisms and their potential roles in nutrient sensing and metabolic regulation in fish.