Engagement of toll-like receptor (TLR)4 ligand, lipopolysaccharide (LPS) with TLR4 in mammals activates two downstream intracellular signaling routes; the myeloid differentiation primary response gene (MyD)88 dependent and independent pathways. However, existence of the later pathway leading to production of type I interferons (IFNs) in avian species has been debated due to conflicting observations. The objective of our study was to investigate whether LPS induces type I IFN production in chicken macrophages leading to antiviral response attributable to type I IFN. We found that LPS elicits type I IFN response dominated by IFN-β production. We also found that reduction in infectious laryngotracheitis virus (ILTV) replication by LPS-mediated antiviral response is attributable to type I IFNs in addition to nitric oxide (NO). Our findings imply that LPS elicits both MyD88 dependent and independent pathways in chicken macrophages consequently eliciting anti-ILTV response attributable to production of both type I IFNs and NO.