Toll-like receptors (TLRs) recognize microbial components and initiate the innate immune responses that control microbial infections. The interaction between ligands of TLR3 and TLR9, poly I:C (an analog of viral double-stranded RNA) and CpG-ODN (a CpG-motif containing oligodeoxydinucleotide) on the inflammatory immune responses, including the production of nitric oxide (NO) and the expression of inducible NO synthase (iNOS), pro-inflammatory cytokines interleukin (IL)-1β and IL-6, and chemokines IL-8 and macrophage inflammatory protein (MIP)-1β, were investigated in chicken monocytes. The NO production was significantly higher when stimulated with a combination of CpG-ODN and poly I:C than with either CpG-ODN or poly I:C alone. Similarly, a significant synergistic effect by CpG-ODN and poly I:C was observed in the up-regulation of iNOS and IL-8 mRNA after 2 h and persisted up to 24 h. Although the combinatory treatment of CpG-ODN and poly I:C enhanced the expression of IL-1β, IL-6, and MIP-1β(3 after 2 h stimulation, the synergism in the up-regulation of IL-1β and IL-6 mRNA was observed after 8-h and 24-h stimulation, respectively, whereas there was no synergistic effect on MIP-1β. Our results demonstrate that CpG-ODN synergizes with poly I:C to induce pro-inflammatory immune response in chicken monocytes.