Mutation analysis in breast cancer has failed to explain the inactivation of RhoBTB2, a candidate breast cancer tumor suppressor gene on chromosome 8p. Some breast cancer-related genes in this region become inactivated by hypermethylation, and hypermethylation of RhoBTB2 abrogates its expression in bladder cancers. The aim of the present study was to determine whether RhoBTB2 was silenced by methylation in breast cancer. Nested methylation-specific PCR (nMSP) and quantitative reverse transcription PCR were used to analyze the methylation status and mRNA levels of RhoBTB2 in 50 paired breast cancer and normal tissues and the results were correlated with clinicopathological characteristics. Promoter methylation and the downregulation of RhoBTB2 mRNA was observed in tumor tissues (P<0.001). mRNA levels were decreased in samples with methylation (χ² = 15.751, P<0.001). RhoBTB2 methylation was observed preferentially in progesterone receptor (PR)-negative samples (P<0.05). The results demonstrated that aberrant methylation of RhoBTB2 may be responsible for the suppression of RhoBTB2 mRNA expression in breast cancer, a significant event during the genesis of breast cancer that correlated with PR status.