Fatty acids serve vital functions as sources of energy, building materials for cellular structures, and 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 time (≥8 h)-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 G protein-coupled receptor 40 (GPR40; also known as free fatty acid receptor-1) protein expression and glucose production that was blocked by GPR40-specific small interfering RNA. Linoleic acid increased intracellular calcium concentration, 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 phospholipase A₂ (cPLA₂) phosphorylation and the release of [³H]-labeled arachidonic acid. 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 proliferator-activated receptor (PPAR) expression, and MK-886 (PPAR-α antagonist) and GW-9662 (PPAR-δ antagonist) inhibited glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. In addition, linoleic acid-induced glucose production was blocked by inhibition of extracellular and intracellular calcium, cPLA₂, COX-2, or PPAR pathways. In conclusion, linoleic acid promoted glucose production via Ca²⁺/PLC, cPLA₂/COX-2, and PPAR pathways through GPR40 in primary cultured chicken hepatocytes.