Vitamin D has predominantly been associated with calcium homeostasis, but recently been associated with several infectious diseases. The active vitamin D metabolite, 1,25-dihydroxyvitamin D₃ (1,25(OH) ₂D₃), is known to regulate the expression of numerous genes throughout the body. 1,25(OH)₂D₃ is synthesized from 25-hydroxyvitamin D₃ by the enzyme 1α-hydroxylase (1α-OHase; CYP27B1). In the endocrine system, 1á-OHase expression, and subsequently 1,25(OH)₂D₃ synthesis, is tightly regulated in the kidney in response to calcium homeostasis. 1,25(OH)₂D₃ produced in the kidney acts systemically to regulate the expression of genes related to calcium homeostasis. In contrast, we have found that bovine monocytes (precursors to macrophages) express 1α-OHase upon activation by toll-like receptor recognition of pathogen associated molecular patterns. Furthermore, we found that 1α-OHase is highly expressed in CD14⁺ cells (monocytes and macrophages) from peripheral blood mononuclear cell cultures stimulated with a purified protein derivative of Mycobacteria bovis and from the infected mammary glands of cows with mastitis. Production of 1,25(OH)₂D₃ by 1α-OHase in bovine monocytes up-regulated the expression of the genes for iNOS and the chemokine RANTES in monocytes. Up-regulation of iNOS resulted in increased production of nitric oxide, a molecule that is considered to have several important roles in immune function. In stimulated peripheral blood mononuclear cell cultures, IFN-γ, IL-17A, and IL-17F, pro-inflammatory cytokines associated with T-cells, were down-regulated by 1,25(OH)₂D₃ that was presumably produced by monocytes. Finally, regulation of gene expression by 1,25(OH)₂D₃ produced by 1α-OHase in monocytes depended on the concentration of 25(OH)D ₃. The circulating concentration of 25(OH)D₃ is controlled by dietary intake of vitamin D₃ and sunlight exposure, so dietary intake of vitamin D₃ and sunlight exposure may impact immune function in cattle. Current requirements for vitamin D in cattle are based on the regulation of calcium homeostasis, but those requirements may be inadequate for proper immune function. Therefore, further investigation is needed to determine vitamin D requirements for proper immune function in cattle.