Mycobacterium bovis, the causative agent of bovine tuberculosis (TB), is a successful pathogen that remains an important global threat to livestock. Cattle naturally exposed to M. bovis normally become reactive to the M. bovis-purified protein derivative (tuberculin) skin test; however, some individuals remain negative, suggesting that they may be resistant to infection. To better understand host innate resistance to infection, 26 cattle from herds with a long history of high TB prevalence were included in this study. We investigated the bactericidal activity, the production of reactive oxygen and nitrogen species and the TB-related gene expression profile after in vitro M. bovis challenge of monocyte-derived macrophages from cattle with TB (n=17) and from non-infected, exposed cattle (in-contacts, n=9). The disease status was established based on the tuberculin skin test and blood interferon-gamma test responses, the presence of visible lesions at inspection on abattoirs and the histopathology and culture of M. bovis. Although macrophages from TB-infected cattle enabled M. bovis replication, macrophages from healthy, exposed cattle had twofold lower bacterial loads, overproduced nitric oxide and had lower interleukin (IL)-10 gene expression (P⩽0.05). Higher mRNA expression levels of inducible nitric oxide synthase, C-C motif chemokine ligand 2 and IL-12 were observed in macrophages from all in-contact cattle than in macrophages from their TB-infected counterparts, which expressed more tumour necrosis factor-α; however, the differences were not statistically significant owing to individual variation. These results confirm that macrophage bactericidal responses have a crucial role in innate resistance to M. bovis infection in cattle.