Culture filtrate protein (CFP) vaccines have been shown to be effective in small animal models for protecting against tuberculosis while immunisation with these types of vaccines in cattle has been less successful. A study was conducted in cattle to evaluate the ability of selected adjuvants and immunomodulators to stimulate protective immune responses to tuberculosis in animals vaccinated with Mycobacterium bovis CFP. Seven groups of cattle (n = 5) were vaccinated with M. bovis CFP formulated with either Emulsigen™ or Polygen™ adjuvant alone or in combination with a specific oligodeoxynucleotides (ODN), polyinosinic acid: polycytidylic acid (poly I:C) or poly I:C and recombinant granulocyte-macrophage colony stimulating factor. Two additional groups were vaccinated subcutaneously with BCG or non-vaccinated. In contrast to the strong interferon-γ (IFN-γ) responses induced by BCG, the CFP vaccines induced strong antibody responses but weak IFN-γ responses. The addition of CpG ODN to CFP significantly enhanced cell-mediated responses and elevated antibody responses to mycobacterial antigens. Of the CFP vaccinated groups, the strongest IFN-γ responses to CFP vaccines were measured in animals vaccinated with CFP/Emulsigen + CpG or CFP/Polygen + CpG. The animals in these two groups, together with those in the BCG and non-vaccinated groups were challenged intratracheally with virulent M. bovis at 13 weeks after the first vaccination and protection was assessed, by examination for presence of tuberculous lesions in the lungs and lymph nodes, 13 weeks later at postmortem. While BCG gave the best overall protection against tuberculosis, significant protection was also seen in animals vaccinated with CFP/Emulsigen + CpG. These results establish an important role for CpG ODN in stimulating protective Th1 responses to tuberculosis in cattle and indicate that a sub-unit protein vaccine can protect these animals against tuberculosis.