Technical Abstract: The active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is an important regulator of immune function. The enzyme that synthesizes 1,25(OH)2D3 from 25-hydroxyvitamin D3 is 1alpha-hydroxylase (1alpha-OHase; CYP27B1). Several in vitro studies have shown that TLR signaling induces expression of 1alpha-OHase in monocytes and macrophages. Synthesis of 1,25(OH)2D3 in monocytes and macrophages presumably allows for local control of 1,25(OH)2D3-mediated gene expression. However, induction of 1alpha-OHase expression at the site of an infection has not been shown. Using Streptococcus uberis mastitis in cattle as a model of bacterial infection, we measured 1alpha-OHase gene expression in mammary tissue and cells isolated from the milk during mastitis. In tissue and secreted cells from the infected mammary glands, 1apha-OHase gene expression was much higher compared to expression in tissue and cells from the healthy glands. Separation of the cells by FACS, revealed that 1alpha-OHase was predominantly expressed in the CD14+ cells from the infected gland. Finally, 24-hydroxylase, inducible nitric oxide synthase (iNOS), and RANTES (regulated upon activation, normally T-cell expressed and secreted; CCL5), genes that are upregulated by 1,25(OH)2D3 in cattle, were expressed significantly more in tissue and cells from the infected glands than from the healthy glands. In conclusion, 1alpha-OHase is upregulated in CD14+ cells in an infected mammary gland during mastitis and seems to provide 1,25(OH)2D3 for local regulation of 1,25(OH)2D3-mediated immune responses. To our knowledge, this study provides the first in vivo evidence that 1alpha-OHase expression is upregulated in macrophages in response bacterial infection.