Escherichia coli infection of the respiratory system in chickens occurs as a sequel to a variety of environmental stressors or microbial infections, culminating as chronic respiratory disease (CRD) syndrome or colibacillosis. These diseases cause significant production losses in poultry. With the growing concerns about the use of antibiotics in animal production, for diseases such as CRD, alternative natural agents, like cytokines, may be considered for enhancing health by stimulating the immune system. The current study was aimed at understanding the in vivo effects of recombinant chicken interferon-gamma (ChIFN-gamma) treatment on a variety of immunologic parameters during E. coli infection in chickens. Administration of ChIFN-gamma to chickens increased the percentage of phagocytes in lung and blood of E. coli-infected birds. At the phenotypic level, there was an increase in the percentage of cells expressing MHC II in the air sac, with a concomitant reduction in the proportion of these cells in blood. Furthermore, the blood plasma from ChIFN-gamma-treated infected birds showed an increased level of interleukin-6 (IL-6) activity. Cumulatively, these findings are indicative of in vivo enhancement of immune responses due to ChIFN-gamma. However, administration of ChIFN-gamma protein did not mitigate the development of air sac lesions following E. coli infection.