Fatty acids serve vital functions as sources of energy, building materials for cellular structures, modulators of physiological responses. Therefore, this study examined the effect of linoleic acid on glucose production and its related signal pathways in primary cultured chicken hepatocytes. Linoleic acid (double-unsaturated, long chain) increased glucose production in a dose ( 10^-4 M)- and a time ( 8 hr)-dependent manner. Both oleic acid (monounsaturated, long chain) and palmitic acid (saturated, long chain) also increased glucose production, whereas caproic acid (saturated, short chain) failed to increase glucose production. Linoleic acid increased GPR40 (free fatty acid receptor 1; FFAR1) protein expression and glucose production which was blocked by GPR40-specific siRNA. Linoleic acid increased [Ca²⁺]_i, which was blocked by EGTA (extracellular calcium chelator)/BAPTA-AM (intracellular calcium chelator), U 73122 (phospholipase C inhibitor), nifedipine, or methoxyverapamil (L-type calcium channel blockers). Linoleic acid increased cytosolic phospholiase A₂ (cPLA₂) phosphorylation and the release of [³H]-labeled arachidonic acid (AA). Moreover, linoleic acid increased the level of cyclooxygenase-2 (COX-2) protein expression, which stimulated the synthesis of prostaglandin E₂ (PGE₂). The increase in PGE2 production subsequently stimulated peroxisome proliferators-activated receptors (PPARs) expression, and MK 886 (PPAR antagonist) and GW 9662 (PPAR antagonist) inhibited glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). In addition, linoleic acid-induced glucose production was blocked by inhibition of extracellular and intracellular calcium, cPLA₂, COX-2, or PPARs pathways. In conclusion, linoleic acid promoted glucose production via Ca²⁺/PLC, cPLA₂/COX-2, and PPARs pathways through GPR40 in primary cultured chicken hepatocytes.