Bovine β-defensin peptides exhibit antimicrobial properties against bacterial pathogens, and expression of several β-defensin genes has been reported in the udder during mastitis. In cattle, toll-like receptor (TLR) and vitamin D signaling pathways induce expression of multiple β-defensin genes; however, β-defensin expression in mammary epithelial cells is only activated via the TLR pathway and is much lower compared with neutrophils and macrophages. The β-defensin gene cluster on bovine chromosome 27 contains multiple potential DNA methylation sites, suggesting the hypothesis that epigenetic mechanisms contribute to induction of the β-defensin response in cattle. The objective of this study was to investigate the effects of DNA methylation and histone deacetylation inhibitors, 5-aza-2'-deoxycytidine (5-Aza) and Trichostatin A (TSA), respectively, on TLR and 1,25-dihydroxyvitamin D₃ (1,25D)-induced expression of β-defensins in primary bovine mammary epithelial cells (bMEC). Primary bMEC cultures obtained via mammary biopsy were treated with 5-Aza (1uM, 72 h), TSA (80 nM, 16 h) or control in combination with lipopolysaccharide (LPS; 100 ng/mL; 16 h) and 1,25D (10 nM, 16 h). Expression of β-defensin 3 (BNBD3), BNBD4, BNBD7, BNBD10, and lingual antimicrobial peptide (LAP) genes was determined using qPCR. The 5-Aza treatment resulted in a > 10-fold increased expression of each gene (P < 0.001). There was also an interaction between LPS and 5-Aza for expression of BNBD3, BNBD10, and LAP genes (P < 0.001). The TSA treatment also increased expression of the BNBD7, BNBD10, and LAP genes approximately 5-fold compared with control (P < 0.01), and there was TSA by LPS interaction for each of the genes (P < 0.05). In contrast, there were not interactions between 1,25D and 5-Aza or TSA for any of the β-defensin genes. These data suggest that DNA methylation and histone acetylation both contribute to β-defensin expression, and that epigenetic mechanisms may affect TLR activation of the β-defensin antimicrobial response of mammary epithelial cells.