The present study was conducted to investigate the effects of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and a combination of both at a ratio of 2:1 on the signalling pathways in lipopolysaccharide (LPS)-stimulated intestinal B lymphocytes from broiler chickens. The results showed that these treatments reduced the production of prostaglandin E2 (PGE 2 ), cyclooxygenase 2 (COX-2), and phospholipase C (PLC) and increased the concentration of inositol triphosphate (IP 3 ) significantly compared with the control group (P < .05). Compared with EPA or DHA alone, the combination of both (2:1) reduced the production PGE 2 , COX-2, and PLC and increased the concentration of IP 3 (P < .05). Therefore, EPA and DHA suppressed the LPSstimulated proliferation of B lymphocytes by interfering with phosphatidylinositol signalling and the second messenger pathways.