Setting: 1,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) is a potent modulator of immune responses and may be beneficial in the treatment of tuberculosis. Recent evidence suggest that 1,25(OH)₂D₃ may affect T-dependent responses in cattle; however, mechanisms by which this vitamin modulates activation of bovine T cells are unclear. Objective: Determine the effects of 1,25(OH)₂D₃ on the expression of CD25, CD44, and CD62L by bovine T cell subsets proliferating in response to antigen stimulation. Design: Antigen-specific recall responses of Mycobacterium bovis bacille Calmette–Guerin (BCG) vaccinated cattle were used as a model system to evaluate effects of 1,25(OH)₂D₃ on the proliferation and activation of bovine T cell subsets. Results: CD4⁺ and γδ TCR⁺ cells were the predominant T cell subsets responding to soluble crude M. bovis-derived antigens (i.e., purified protein derivative and a BCG whole cell sonicate) by proliferation and activation-induced alterations in phenotype. These subsets exhibited increased CD25 and CD44 mean fluorescence intensity (mfi) and decreased CD62L mfi upon antigen stimulation. Addition of 1,25(OH)₂D₃ inhibited proliferation of CD4⁺ cells and decreased the expression of CD44 on responding (i.e., proliferating) CD4⁺ and γδ TCR⁺ cells. Conclusion: These findings suggest that the production of 1,25(OH)₂D₃ by macrophages within tuberculous lesions would inhibit proliferation and CD44 expression by co-localized CD4⁺ and γδ TCR⁺ cells.