Background Defensins are antimicrobial peptides composed of three conserved disulfide bridges, a β-sheet, and both hydrophobic and cationic amino acids. In this study, we aimed to demonstrate the immunomodulation role of avian β-defensin 8 (AvBD8) in a chicken macrophage cell line.Results Chicken AvBD8 stimulated the expression of proinflammatory cytokines (interleukin (IL)-1β, interferon-γ, and IL-12p40) and chemokines (CCL4, CXCL13, and CCL20) in macrophages. Furthermore, by western blotting and immunocytochemistry, we confirmed that AvBD8 activated the mitogen-activated protein kinase (MAPK) signaling pathway via extracellular regulated kinases 1/2 (ERK1/2) and p38 signaling molecules.Conclusion Overall, AvBD8 plays a crucial role in host defense as not only an antimicrobial peptide, but also an immunomodulator by activating the MAPK signaling pathway and inducing the expression of proinflammatory cytokines and chemokines.