Infectious bursal disease virus (IBDV) infects and replicates in the dividing B lymphocytes of chickens. In the present study, the in vivo effect of IBDV infection on chicken macrophage populations and macrophage activation were examined. Specific-pathogen-free chickens were exposed to virulent IBDV and splenic macrophages were recovered during the acute phase of the disease. At 3 and 5 days post-infection (dpi), spleens of virus-exposed chickens had fewer macrophages than those of virus-free controls (p < 0.05). Confocal microscopic examination revealed cells that were positive for both KUL01 (macrophage surface marker) and R63 (IBDV-VP2), indicating presence of the virus in macrophages. MQ-NCSU cells, an avian macrophage cell line, were susceptible to replication of IBDV. In addition, splenic macrophages were activated and had temporarily increased levels of mRNA transcripts of pro-inflammatory mediators, including IL-1β, IL-6, IL-18, and iNOS. The robust expression of proinflammatory cytokine transcripts, along with a decrease in macrophage numbers, suggest that IBDV activates and may lead to a reduction of resident macrophages in vivo.