Various approaches have been developed to improve the efficacy of DNA vaccination, such as the use of plasmids expressing cytokines as molecular adjuvants. The purpose of the present study was to determine whether co-administration of a plasmid containing a chicken granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and a plasmid containing the S1 gene of infectious bronchitis virus (IBV) could enhance the immune response and protection efficacy in chickens against challenge by virulent IBV. Plasmids carrying the S1 gene of IBV (pVAX-S1) and the chicken GM-CSF gene (pVAX-chGM-CSF) were constructed. Seven-day-old chickens were injected intramuscularly with pVAX-S1, pVAX-chGM-CSF, or both and boosted 2 weeks later. Chickens were challenged with virulent IBV at 3 weeks after the booster immunization and observed for 2 weeks. The results showed that co-administration of pVAX-chGM-CSF led to a significant enhancement of humoral and cellular responses over that of vaccination with pVAX-S1 alone. In addition, vaccination with pVAX-chGM-CSF and pVAX-S1 provided 86.7% protection (13/15) against IBV challenge. In contrast, only 73.3% of the chickens were protected against IBV challenge by pVAX-S1 vaccination alone. These results strongly indicate that chGM-CSF can be used as a molecular adjuvant to enhance the protective immunity induced by an IBV-specific DNA vaccine.