ABSTRACT: Host defense peptides (HDPs) are multifunctional immune molecules that respond to bacterial and viral pathogens. In the present study, bone marrow-derived cells (BMCs) and chicken embryonic fibroblasts (CEFs) were cultured from a Leghorn line (Ghs6) and Fayoumi line (M15.2), which are inbred chicken lines relatively susceptible and resistant to various diseases, respectively. The cells were treated by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly(I:C)) and, subsequently, mRNA expression of 20 chicken HDPs was analyzed before and after the stimulation. At homeostasis, many genes differed between the chicken lines, with the Fayoumi line having significantly higher expression (p < 0.05) than the Leghorn line: AvBD1, 2, 3, 4, 6, and 7 in BMCs; CATH1, CATH3, and GNLY in CEFs; and AvDB5, 8, 9, 10, 11, 12, 13 in both BMCs and CEFs. After LPS treatment, the expression of AvBD1, 2, 3, 4, 5, 9, 12, CATH1, and CATHB1 was significantly upregulated in BMCs, but no genes changed expression in CEFs. After poly(I:C) treatment, AvBD2, 11, 12, 13, CATHB1 and LEAP2 increased in both cell types; CATH2 only increased in BMCs; and AvBD3, 6, 9, 14, CATH1, CATH3, and GNLY only increased in CEFs. In addition, AvBD7, AvBD14, CATH1, CATH2, GNLY, and LEAP2 showed line-specific expression dependent upon cell type (BMC and CEF) and stimulant (LPS and poly(I:C)). The characterization of mRNA expression patterns of chicken HDPs in the present study suggests that their functions may be associated with multiple types of disease resistance in chickens.