OBJECTIVE To characterize activation and expression of immune genes of chicken macrophages after in vitro stimulation with lipopolysaccharide (LPS) and mouse erythrocytes. ANIMALS Five 15-day-old chickens and 2 BALB/c mice. PROCEDURES Macrophages were extracted from chicken bone marrow or peripheral blood and then stimulated with cytokines secreted from cell lines L929 and HD11. Stimulated chicken macrophages were further cocultured with LPS or mouse erythrocytes, and gene transcription of some distinctive cytokines was detected by use of a real-time PCR assay. RESULTS Morphological features and phagocytic function of macrophages were characterized. Activated macrophages had an elongated shape with a large cell nucleus, and they had phagocytic function. Distinctive genes encoding the surface marker gene CD11b were identified; high quantities of CD11b were transcribed. Relative transcription of chicken genes BF and BL in mature cells cocultured with both stimuli was lower than for control cells. However, the quantity of genes encoding M1- or M2-distinctive cytokines (interleukin [IL]-1β, IL-10, IL-12, inducible nitric oxide synthase, tumor necrosis factor-α, and transforming growth factor-β) that were transcribed differed significantly between stimulation with LPS and mouse erythrocytes. CONCLUSIONS AND CLINICAL RELEVANCE Chicken macrophages were differentially stimulated by LPS and mouse erythrocytes, which suggested that in vitro stimulation can distinctly influence the transcription and expression of immune genes of chicken macrophages.