Objective To analyze the expressions of COX-2, p-ERα and CYP1B1 in human breast cancer tissues and ERα-positive human breast cancer cell line MCF-7, and to investigate the influence of 1,25-dihydroxyvitamin D3 \[1,25(OH)2D3\] on proliferation, cell cycle transformation, and CYP1B1 protein expression in MCF-7 cells. Methods Immunohistochemical method was applied to examine the expressions of COX-2, p-ERα and CYP1B1 protein in 42 breast cancer tissues, and their association was analyzed. The effects of 1,25(OH)2D3 on MCF-7 cell proliferation was investigated by MTT assay and the optimal concentration of 1,25(OH)2D3 was determined for the following experiment. The cell cycle was analyzed by flow cytometry and COX-2 mRNA expression in MCF-7 cells was measured by RT-PCR. PGE2 level was detected by ELISA in the culture supernatant. The expression of COX-2, p-ERK, p-ERα and CYP1B1 protein was determined by Western blotting analysis and the distribution of COX-2, p-ERα and CYP1B1 expression in MCF-7 cells was examined by immune cell fluorescence. Results COX-2, p-ERα and CYP1B1 protein were positive in human breast cancer tissues and their expressions were positively correlated with each other(P<0.05).1,25(OH)2D3 inhibited the proliferation of MCF-7 cells in a time- and dose-dependent manner (P<0.05). Treatment with 100 nmol/L 1,25(OH)2D3 for 72 h significantly arrested cell cycle in G0/G1 phase (P<0.05), decreased the expression of COX-2 mRNA in MCF-7 cells (P<0.05), decreased PGE2 level in the cell culture supernatant (P<0.01), and down-regulated p-ERK, p-ERα and CYP1B1 protein expression(P<0.05). Conclusion COX-2/PGE2 pathway plays a positive role in regulating CYP1B1 expression in breast cancer.1,25(OH)2D3 may inhibit the growth of MCF-7 cells and down-regulate CYP1B1 through COX-2/PGE2 pathway.