Postpartum hypocalcemia has been associated with suppressed neutrophil and lymphocyte function in dairy cattle. It was hypothesized that mitigation of postpartum hypocalcemia would enhance activation of host-defense mechanisms during the postpartum period. The objective of this study was to determine the effects of pre-partum DCAD in combination with vitamin D supplementation on expression of a cluster of β-defensin (DEFB) host-defense genes. Pregnant dry Holstein cows (n = 51, multiparous) of 255 d gestation were blocked by parity and randomly assigned to 4 prepartum diets of positive (+130 mEq/kg) or negative (−130 mEq/kg) DCAD and either 3 mg vitamin D₃ or 3 mg of 25-hydroxyvitamin D₃ (25D) per 11 kg of DM/d. Treatment diets were fed for the last 21 d of gestation, and, upon calving, cows were fed the same lactation ration for the first 49 DIM. Peripheral blood neutrophils and monocyte cultures were collected at d 0 and d 3 of calving and stimulated with 75 ng/mL of 25D and a combination of 100ng/mL of lipopolysaccharide (LPS) and 75 ng/mL of 25D. The mRNA transcripts of DEFB genes were quantified by real-time PCR, and data were analyzed with a general linear mixed model to test for fixed effects and interactions of experimental variables. Neutrophil gene expression of DEFB4 (P ≤ 0.05), DEFB5 (P ≤ 0.05), DEFB7 (P = 0.1), and DEF10 (P = 0.1) was greater in cows fed the negative DCAD. Neutrophils had lower expression of DEFB3, DEFB4, DEFB7, DEFB10, and lingual antimicrobial peptide in response to LPS at d 3 of calving compared with d 0, (P ≤ 0.05). Cows supplemented with vitamin D₃ showed greater DEFB3, DEFB4, DEFB5, DEFB7, and DEF10 gene expression in monocytes (P ≤ 0.05) than 25D supplemented cows. In conclusion, pre-partum DCAD and 25D affect neutrophil and monocyte β-defensin responses and have implications for improving transition cow immunity.