Mycobacterium bovis, the classical causative agent of bovine tuberculosis (BTB), infects animals of agricultural importance and other mammals, including humans. Neutrophils are one of the first lines of defense against all microbes and produce a diverse collection of antimicrobial molecules, which play an important role in the early control of tuberculosis progression. An interferon (IFN)-inducible neutrophil-driven blood transcriptional signature that consisted of both IFN-γ and type I IFN-α/β signaling has been identified in human tuberculosis, supporting a role for neutrophils in the pathogenesis of tuberculosis disease. However, it is unknown whether bovine neutrophils play a similar role during M. bovis infection. Thus, we assessed the expression levels of ten IFN-inducible transcriptional genes in neutrophils from healthy cattle stimulated by M. bovis and neutrophils isolated from three groups of cattle of different infection status, and in addition, examined the changes in the expression of myeloperoxidase (MPO) and pentraxin-related protein pentraxin-inducible protein (PTX3) genes during bovine tuberculosis infection. Our results demonstrated a specific expression pattern of IFN-inducible transcriptional genes and MPO and PTX3 genes in neutrophils during bovine tuberculosis infection. The observed expression pattern provides a potential diagnostic tool, which may have implications for vaccine and therapeutic development to combat the bovine tuberculosis epidemic.