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Vitamin D signaling in response to pathogen-associated molecules contributes to activation of innate immune responses of bovine monocytes. We hypothesized that lipopolysaccharide (LPS) of bacteria associated with mastitis in dairy cows activates the vitamin D pathway in innate immune cells of the udder and that increasing availability of 25-hydroxyvitamin D3 [25(OH)D3] would augment expression of vitamin D-associated genes. The objective of this experiment was to determine the effects of intramammary LPS and 25(OH)D3 treatments on activation of the vitamin D pathway and innate immune responses of mammary immune cells. Individual mammary quarters of 5 lactating cows were treated with placebo control, 100 μg of 25(OH)D3, 5 μg of LPS, or a combination of 100 μg of 25(OH)D3 and 5 μg of LPS. Somatic cells from milk were evaluated for percentage of neutrophil and macrophage populations and expression of genes associated with vitamin D metabolism and innate immunity. Data from samples collected from 4 to 12 h after challenge were analyzed for main effects of LPS and 25(OH)D3 treatments, treatment interactions, and simple effects of 25(OH)D3 treatment. Data from samples collected at the time of challenge were used as covariates. The percentages of neutrophils in milk at 8 h postchallenge were 58 ± 10, 82 ± 11, 89 ± 10, and 63 ± 10% of total cells in milk from control, 25(OH)D3, LPS, and LPS plus 25(OH)D3 glands, respectively, such that the interaction of LPS and 25(OH)D3 was significant. Expression of the vitamin D 1α-hydroxylase (CYP27B1) and vitamin D receptor genes was upregulated by LPS treatment in total cells, macrophages, and neutrophils in milk. In addition, expression of the vitamin D 24-hydroxylase (CYP24A1) gene in milk somatic cells was upregulated by 25(OH)D3 and LPS treatments. The inducible nitric oxide synthase (iNOS), chemokine (C-C-motif) ligand 5 (CCL5), β-defensin 3 (DEFB3), DEFB7, and DEFB10 genes were upregulated by LPS treatment in total cells and neutrophils from milk. Expression of iNOS in milk somatic cells tended to be affected by the interaction between LPS and 25(OH)D3, such that 25(OH)D3 tended to increase iNOS in the absence of LPS but not in the presence of LPS. Furthermore, expression of CCL5 in macrophages was downregulated by 25(OH)D3. In conclusion, intramammary endotoxin challenge activates the vitamin D pathway in mammary macrophages and neutrophils, and intramammary 25(OH)D3 treatment alters the percentage of neutrophils and expression of immune genes in milk somatic cells. [ABSTRACT FROM AUTHOR]