In the present study, calves were infected with Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium avium subsp. avium (M. avium), Mycobacterium kansasii (M. kansasii), or Mycobacterium bovis (M. bovis) to determine differences in cellular immunity. Comparative cellular responses were assessed upon stimulation of cells with mycobacterial whole cell sonicates respective of each infection group. Antigen-specific whole blood interferon gamma (IFN-γ) responses were observed in all infection groups compared to noninfected control calves, however, responses were more robust for M. bovis calves. Upon antigen stimulation of PBMCs, secretion of IFN-γ and IL-10 was higher for M. bovis calves compared to other infection groups. In contrast, IL-12 secretion was lower for M. bovis calves compared to MAP infected calves. Within the total PBMC population, higher numbers of CD4+, CD8+, and γδ TCR + T cells were observed for MAP and M. avium calves compared to M. bovis calves. This aligned with higher expression of CD26 on these subpopulations for MAP and M. avium calves, as well. In contrast, greater expression of CD25 was observed on CD4+ and γδ TCR + T cells and natural killer cells for M. bovis calves. Overall, similarities in cellular immune responses were observed between the closely related MAP and M. avium during infection of calves. In contrast, significant differences were noted between calves infected with MAP and M. bovis. This suggests that host immune responses to different mycobacteria may impact interpretation of diagnostic tools based upon their cellular immunity.