In this study, we differentiated murine bone marrow-derived macrophages (BMDMs) into M0, M1, and M2 in the presence or absence of calcitriol. Real-time PCR analysis of gene expression, FACS analysis of surface markers, and chemokine/cytokine production assays were performed. In addition, the effect of the conditioned media (CM) from murine breast cancer 4T1 (metastatic) and 67NR (non-metastatic) and Eph4-Ev (normal) cells with and without calcitriol on the polarization of M1/M2 cells was determined. We found that calcitriol enhanced the differentiation of M2 macrophages, which was manifested by increased expression of Cd206 and Spp1 mRNA and CD36, Arg, and CCL2 in M2 BMDMs and by decreased expression of Cd80 and Spp1 mRNA and IL-1, IL-6, OPN, and iNOS in M1 BMDMs. 4T1 CM showed a higher effect on the gene and protein expression in macrophages than 67NR and Eph4-Ev, with the greatest effect observed on M2 macrophages which increased their differentiation and properties characteristic of alternative macrophages. Moreover, M2 macrophages differentiated with calcitriol-stimulated migration of 4T1 and 67NR cells through fibronectin and collagen type IV, respectively. Overall, our results indicated that vitamin D supplementation may not always be beneficial, especially in relation to cancers causing excessive, pathological activation of the immune system.Keywords:4T1 cells; 67NR cells; BMDMs; M2 macrophages; breast cancer cells; calcitriol; macrophages; vitamin D.