A number of studies have demonstrated significant protection of cattle against bovine tuberculosis following vaccination with the Pasteur strain of Mycobacterium bovis bacille Calmete-Guerin (BCG). However, it is unclear if other daughter strains of BCG are as effective, which is an important issue to resolve for a variety of regulatory compliance issues. This study compared the protective immune responses to bovine tuberculosis induced in cattle vaccinated with BCG Danish with those induced by BCG Pasteur. Groups of calves (n = 10) were vaccinated with 106 colony forming units (CFU) BCG Pasteur prepared from a fresh liquid culture, 106 CFU BCG Danish prepared from a fresh liquid culture or 0.4 mg of reconstituted freeze-dried culture of BCG Danish. Another group (n = 10) served as non-vaccinated controls. BCG Pasteur induced significantly higher and more sustained levels of bovine purified protein derivative (PPD)-specific gamma interferon (IFN-γ) in whole-blood cultures following vaccination compared to either fresh culture BCG Danish or freeze-dried BCG Danish. Vaccination with a fresh culture of BCG Pasteur, fresh culture BCG Danish and freeze-dried BCG Danish gave a significant enhancement in three, four and three pathological and microbiological parameters of protection, respectively, compared to the non-vaccinated group. These results demonstrate the Danish strain of BCG is a viable alternative to BCG Pasteur for vaccination of cattle as both strains had similar efficacy and there was little difference between freshly cultured and freeze-dried formulation of BCG Danish. The results also show that post-vaccination antigen-specific IFN-γ levels in whole blood is not always a reliable indicator of protection against a subsequent virulent challenge.