The study was conducted to investigate the effects of fish oil and maize oil on nuclear factor kappa B (NFκB) gene expression and the downstream pathways of intracellular signaling in spleen of chickens after lipopolysaccharide (LPS) stimulation. Two hundred eighty eight chickens were assigned in a 2×2 factorial design. Factors were dietary fat type (4.5% maize oil or 4.5% fish oil) and immunological stimulation (LPS or saline). LPS increased levels of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) of spleen in chickens after the second LPS stimulation on 28 d of age. Fish oil alleviated the increase of EPA and DHA in spleen of chickens after LPS stimulation at 27 d of age. Fish oil supplementation decreased prostaglandin 2 (PGE₂) production and the activity of cyclooxygenase 2 (COX₂) after LPS stimulation. LPS stimulation increased the activity of phospholipase C (PLC) in spleen of chickens. And fish oil inhibited activity of PLC in spleen of chickens stimulated by LPS. Meanwhile fish oil decreased the production of inositol triphosphate (IP₃) in spleen of chickens stimulated by LPS. Fish oil alleviated the mRNA abundance elevation of nuclear factor kappa B (NFκB) after LPS stimulation. These results showed that fish oil down-regulated the production of IP₃ and PGE₂ through inhibiting the activity of PLC and COX₂ in spleen of chickens, respectively. The results of NFκB gene expression suggested fish oil might alleviate immune stress at the level of transcription.