In this study, ketoconazole was found to selectively inhibit ERβ with IC50 value of 0.79 μM and showed good antiproliferative activity on human MDA-MB-231 breast cancer cell line with IC50 value of 8.95 μM, which indicated that ketoconazole may have more broad-spectrum anti-cancer indications with therapeutic effects of breast cancer in clinic. This evidence concerns the gene ESR2 and cancer.