INTRODUCTION: Dendritic cells (DCs) play a crucial role in orchestrating immune responses by bridging innate and adaptive immunity. METHODS: The present study takes advantage of newly available chicken immunological tools to further characterize chicken monocyte-derived dendritic cells (MoDCs), focusing on their phenotype, and functions, including antigen capture and T-cell stimulation, and response to live Newcastle disease virus (NDV) stimulation. RESULTS: Adherent chicken PBMCs were cultured with recombinant chicken granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), for 5 days, leading to the upregulation of putative CD11c and MHCII, markers of DC differentiation. Subsequent stimulation with lipopolysaccharide (LPS) or 24 h triggered phenotypic maturation of MoDCs, characterized by the increased surface expression of MHCII and co-stimulatory molecules CD80 and CD40, and elevated IL-12p40 secretion. This maturation reduced endocytic capacity but enhanced the allogenic stimulatory activity of the chicken MoDCs. Upon NDV stimulation for 6 h, MoDCs upregulated antiviral pathways, including retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), alongside increased production of type I interferons (IFNs), and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), IL-1β, and IL-6. However, these responses were downregulated after 24 hours. CONCLUSION: These findings provide a comprehensive characterization of chicken MoDCs and suggest their potential as a model for studying host-pathogen interactions.