Objective To investigate the expression level and methylation status of endothelin receptor B (EDNRB) gene in four breast cancer cell lines and the effect of re-expression of methylated EDNRB on MCF-7 cell proliferation. Methods Methylation-specific PCR (MS-PCR) and bisulfite sequencing PCR (BSP) were used to detect methylation status of EDNRB in breast cancer cell lines. EDNRB gene expression level was measured by reverse transcription-PCR (RT-PCR). The colorimetric MTT assay and colony formation were used to investigate the effect of re-expression of methylated EDNRB on MCF-7 cell proliferation. Results No gene expression and promoter hypermethylation of EDNRB were detected in MCF-7 and ZR-75-1 cell lines. While the low frequency of methylation event happened in MDA-MB-231 cells which had the highest expression level of EDNRB. These results suggested that, in 4 breast cancer cell lines, the negative correlation existed between methylation status in the promoter region and expression of EDNRB gene. The expression of EDNRB gene in MCF-7 cells could be recovered by 5-azacytidine (5-Aza-CR) in a dose-dependent manner from 5 μmol/L to 10 μmol/L. Re-expression of EDNRB gene significantly inhibited cell proliferation. Conclusion Methylation of CpG islands in the promoter region of EDNRB gene may play important roles in the carcinogenesis and development of breast cancer. EDNRB gene may be a new molecular marker in the early diagnosis of breast cancer.