Vitamin D deficiency (VDD) is associated with enhanced susceptibility to multiple respiratory diseases in humans, including tuberculosis. However, the consequences of VDD for disease susceptibility in calves are unknown. Previously we developed a model to drive divergent circulating 25OHD concentrations in cattle, where animals were supplemented with vitamin D 3 (vit D 3) from birth to 7 months of age. Calves in the control group (Ctl) received a diet containing a standard vit D 3 concentration, whereas the vit D group (VitD) received a diet with the highest vit D 3 concentration allowed under EU guidelines. Here, we assessed the microbicidal activity and immunoregulatory effect of divergent 25OHD circulating levels to Mycobacterium bovis BCG challenge exvivo. Blood samples from Ctl and VitD calves were taken at 1-, 3-and 7-months of age. 25OHD concentrations were significantly different at 7 months (but not at 1 or 3 months) with animals from the VitD group having higher serum levels. Differences in microbicidal activity followed the same pattern, with no significant differences observed at 1 and 3 months, but at 7 months a significant increase in the percentage of bacteria killed was detected. Furthermore, analysis of the reactive oxygen species (ROS) and nitric oxide (NO) in serum showed a higher production of ROS and NO in VitD-supplemented calves. In contrast, serum concentrations of IL-1β and IL-8 were significantly lower. A similar anti-inflammatory profile was observed after gene expression analysis, with a significant downregulation of a cluster of genes including IL1B, IL1R1, CXCL1, CXCL2, CXCL5, MMP9 and COX2 and an upregulation of CXCR1, CX3CR1 and NCF1, in VitD calves after BCG challenge relative to Ctl animals. Collectively, these results suggest that dietary vit D 3 boosts antimicrobial and innate immune responses and thereby could improve host anti-mycobacterial immunity.