Active vitamin D has several antitumor effects, including prodifferentiative, antiproliferative and proapoptotic functions in a number of tissues via its binding to vitamin D receptor. The 24‑hydroxylase (CYP24A1) and 1‑hydroxylase (CYP27B1) enzymes are considered to be pivotal determinants of the local concentration of active vitamin D. The aim of the present study was to investigate the mRNA expression levels of the CYP24A1 and CYP27B1 genes in malignant and normal breast tissues. The tumor and adjacent normal tissue samples of 30 patients with breast cancer were acquired from the Iran National Tumor Bank, Imam Hospital (Tehran, Iran). RNA was extracted and, following cDNA synthesis, Taq‑Man quantitative polymerase chain reaction analysis was performed for CYP24A1 and CYP27B1. The results demonstrated that the mRNA expression of CYP27B1 was downregulated in the tumor tissues, compared with the adjacent normal tissues (P<0.01), whereas the mRNA expression of CYP24A1 was significantly upregulated in the tumor tissues (P<0.01). This major difference revealed that the normal breast tissues transcriptionally expressed CYP24A1 slightly. These results are suggestive of dysregulation of the vitamin D signaling and metabolic pathways during tumorigenesis in breast cancer. Local alterations in the anabolism and catabolism of active vitamin D in breast cancer by the CYP27B1 and CYP24A1 may impair its anticancer functions.