The bovine innate immune system has a strong repertoire of antimicrobial defenses to rapidly attack infectious pathogens that evade physical barriers of the udder. Exploration of the intracrine vitamin D pathway of bovine macrophages has generated a better understanding of the signals that initiate antimicrobial defenses that protect the udder. In the intracrine vitamin D pathway, pathogen recognition receptors upregulate CYP27B1 mRNA that encodes for the enzyme that converts 25-hydroxyvitamin D (25D) to the active vitamin D hormone, 1,25-dihydroxyvitamin D₃ (1,25D). The 1,25D, in turn, increases nitric oxide and b-defensin antimicrobial responses of bovine macrophages. Investigation of the intracrine vitamin D pathway using experimental models of mastitis of dairy cows has revealed that vitamin D signaling in innate immune cells contributes to defense of the mammary gland. Expression of the CYP27B1 and the vitamin D receptor genes rapidly increased in macrophages and neutrophils of the udder in experimental models of mastitis in dairy cows. Intramammary 1,25D treatment increased expression of inducible nitric oxide synthase and b-defensin genes in immune cells of the mammary gland. Furthermore, Intramammary 25D treatment slowed onset and decreased severity of experimental Streptococcus uberis mastitis. The evidence that vitamin D contributes to defense of the mammary gland provides a path for development for alternative solutions (i.e., nutritional, genetic, therapeutic) to increase mastitis resistance of dairy cows. Continued exploration of the intrinsic cellular pathways that specifically promote antimicrobial defenses of the udder, as demonstrated with the vitamin D pathway, is needed to support mastitis control efforts for dairy cows.