Cytochrome P4501B1 (CYP1B1) is responsible for tumor progression in estrogen receptor-positive breast cancer due to its key role in estrogen metabolism, which is upregulated by PGE2, the main product of COX-2 that is found to be overexpressed in many breast tumors. Previous studies reported that inhibition of the COX-2/PGE2 pathway, by 1,25-dihydroxyvitamin D3 in MCF-7 breast cancer cells. The aim of this study was to investigate if the CYP1B1 protein expression shows covariation with the COX-2 and phosphorylated ERα (p-ERα) in human breast cancer. We also investigated whether 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] downregulates CYP1B1 via the COX-2/PGE2 pathway in MCF-7 cells. We analyzed the expression of COX-2, p-ERα and CYP1B1 using an immunohistochemical staining assay. In the present study, COX-2 was correlated to p-ERα (P<0.001) and CYP1B1 (P=0.001), p-ERα was correlated to CYP1B1 (P=0.012). We assessed the effects of 1,25(OH)2D3 on MCF-7 cells. 1,25(OH)2D3 treatment inhibited MCF-7 cell growth in a time-and dose-dependent manner; the cell cycle was arrested in the G0/G1 phase. Treatment with 100 nmol/l 1,25(OH)2D3 for 72 h significantly decreased the expression of COX-2 mRNA in MCF-7 cells (P<0.05), decreased the levels of PGE2 in cell culture supernatant (P<0.01), and downregul-ated p-ERK, p-ERα and CYP1B1 protein expression (P<0.05). Taken together, these results suggest that the COX-2/PGE2 pathway positively regulates the expression of CYP1B1 in breast cancer. 1,25-Dihydroxyvitamin D3 inhibits the growth of MCF-7 cells and downregulates CYP1B1 mediated by the COX-2/PGE2 pathway.