Synthetic oligodeoxynucleotides (ODN) containing cytosine-phosphodiester-guanine (CpG) motifs (CpG-ODN) have been shown to be effective immunoprotective agents and vaccine adjuvants in a variety of bacterial, viral, and protozoan diseases in different animal species. The objective of this study was to compare the immune response of chickens to a killed Escherichia coli vaccine combined with oil in water emulsion or with CpG-ODN. Birds were vaccinated with killed E. coli antigens with either 10 or 50 μg of CpG-ODN on days 10 and 20 of age. At day 30, a virulent isolate of homologous E. coli was applied on a scratch site on the caudal abdominal region. Birds were examined for 10 days post-E. coli challenge, and pathologic and bacteriologic assessments were conducted on all birds that were either found dead or euthanized. The E. coli vaccine group that received no CpG-ODN had a survival rate of 65%. In contrast, groups that received the vaccine with CpG-ODN adjuvant had significantly higher survival rate of 92% (P < 0.01) with isolation of low numbers of E. coli from internal organs. Total IgG against E. coli antigens was highest in groups that received CpG-ODN as an adjuvant. Birds that received vaccine containing CpG-ODN had minimal inflammatory reaction without tissue necrosis at the injection site. Severe tissue necrosis was present in birds that received vaccine containing oil in water emulsion adjuvant. This study demonstrated that CpG-ODN is an effective vaccine adjuvant in chickens and results in minimal tissue destruction. This study is the first study in which CpG-ODN has been demonstrated to produce an adaptive immune response, at a significant level, against an extracellular bacterial infection in chickens.