1,25-dihydroxyvitamin D3 (1,25(OH)2D3), a bioactive Vitamin D, is known to regulate immune responses in mammals. However, its impact on the innate immune responses of Japanese Black cattle, which are beef cattle endemic to Japan, remains unknown. Thus, in this study, we investigated the effect of 1,25(OH)2D3 on the immune responses of PBMC from Japanese Black cattle. PBMC were cultured with or without 1,25(OH)2D3 for measurement of cell viability, and stimulated with or without 1,25(OH)2D3 and lipopolysaccharide (LPS) for measurement of the gene expressions. As the results, the treatment of 1,25(OH)2D3 increased the cell viability. It also upregulated antibacterial peptides, DEFB10 and LAP with or without LPS stimulation. Moreover, 1,25(OH)2D3 enhanced the inflammatory responses, CXCL8 with LPS stimulation and NOS2 with or without LPS stimulation, while reducing the expression of anti-inflammatory cytokine IL10 with or without LPS stimulation, leading to an inflammatory phenotype. However, in contrast to humans and mice, 1,25(OH)2D3 did not alter the expression of TNF and downregulated TREM1 with LPS treatment. These results suggest that 1,25(OH)2D3 potentiates the innate immune responses of Japanese Black cattle, albeit with different effects and mechanisms as compared to humans and mice.