Bacterial mastitis is a significant problem for the dairy industry. A vaccine against mastitis pathogens could potentially target dendritic cells (DC). While there has been some research describing bovine DC populations in-vitro, little is known about DC in mammary tissue. In this study, immunohistofluorescence was used to identify and localize bovine mammary DC. DC were found in alveoli, in epithelia, and in interalveolar tissue. Fluorescence-activated cell sorting (FACS) was used to characterize mammary DC as expressing CD11c, MHC-II, CD205, CD11b, and CD8α. FACS allowed us to distinguish DC (CD14lo) from macrophages (CD14hi). Two DC subsets, CD11a-, CD11alo, were evident in the mammary gland while an additional CD11ahi population was identified in the supramammary lymph node. After phagocytosis of bacterial components such as lipopolysaccharide (LPS), DC undergo a maturation process, in which they upregulate homing receptors, such as CCR7, and antigen presentation markers, including MHCII and CD80. A primary cell culture model was used to evaluate changes in transcription of CD80 and CCR7 after LPS stimulation. Cell cultures contained digested and Ficoll separated mammary tissue or supramammary lymph node tissue. While the presence of CCR7 and CD80 was confirmed, CD80 and CCR7 transcripts were not upregulated after LPS stimulation. Further, CD11c, CD14, MHCII, CD11b, CD11a, and CD205 protein levels, as assessed by FACS, were similar in LPS stimulated cultures and unstimulated controls. Overall, these studies provide a better understanding of mammary gland immunology, while potentially aiding in the development of novel DC based vaccines.