AIMS:This study aimed to investigate the ability of CpG7909 adjuvant to enhance immunogenicity and protective efficacy of a subunit vaccine composed of ESAT6-Ag85A fusion protein (Pe685a) of Mycobacterium tuberculosis. METHODS AND RESULTS:ELISA was used to detect specific antibody and IFN-γ expression in sera; ELISPOT, to detect IFN-γ expression in splenocytes; MTT assay and FACS, to detect T-lymphocytes proliferation in spleens; and RT-PCR, to detect cytokines expression in lungs of mice after immunization. Bacterial load and histopathological lesions in lungs or spleens of mice challenged with Myco. tuberculosis H37Rv strain were analysed. Compared with incomplete Freund's adjuvant, CpG7909 induced more potent production of Pe685a-specific IgG2a/IgG1 antibody and higher expression of IFN-γ in sera, stimulated more generation of antigen-specific IFN-γ-secreting splenocytes, enhanced frequencies of CD3(+) CD4(+) and CD3(+) CD8(+) T-lymphocytes in spleen and increased transcription of TNF-α, IFN-γ, IL-6 and TLR9 in lung. However, lower bacterial load in lung and less severe lung pathology were not observed in CpG7909 group mice. CONCLUSIONS:CpG7909 is able to enhance immunological effects of Pe685a subunit vaccine, but does not confer significant protective efficacy against Myco. tuberculosis infection. SIGNIFICANCE AND IMPACT OF THE STUDY:CpG7909 as an adjuvant of subunit vaccine against Myco. tuberculosis is worthy of further investigation.