Utilizing RNA interference technology with siRNA in the HD11 macrophage cell line, we determined how the inhibition or knock-down of the iNOS (inducible nitric oxide synthase) gene affected IFN-γ-induced macrophage production of nitric oxide (NO) and mRNA expression of genes involved in this biological pathway in the chicken. Chicken macrophages produce NO when stimulated with recombinant chicken IFN-γ, however, when transfected with iNOS siRNAs, the production of NO is significantly decreased. We observed a 14–28% reduction in NO production by IFN-γ-stimulated HD11 cells at 48 h after initial siRNA transfection compared to non-transfected IFN-γ-stimulated macrophages. Significant knock-down of iNOS mRNA expression (15 to 50-fold lower) was observed for each of four iNOS siRNAs, when compared to non-transfected IFN-γ-stimulated macrophages and to those treated with a negative control siRNA. The IFN-γ-stimulated chicken macrophages transfected with iNOS siRNAs did not show altered levels of mRNA expression for genes involved in IFN-γ signaling and iNOS pathways (IL-1β, IL-6, IFN-γ, TGF-β4, or SOCS-3) suggesting that the observed decrease in NO production is a direct result of siRNA mediated knock-down of iNOS, rather than IFN-γ-induced changes in the other genes tested.