R-Ras, a member of the Ras superfamily, is expressed in a wide variety of tissues and regulates cell adhesion, migration, differentiation and apoptosis. Research has raised the possibility that R-Ras may function as a positive regulator of cell proliferation and transformation in the breast. To understand the possible role of R-Ras in breast epithelial carcinogenesis, the expression and activation of R-Ras were detected in each of 69 pairs of breast cancer tissues and normal tumor-adjacent tissue samples by qRT-PCR, western blot analysis and GST pull down assay; 12 available cell lines were also subjected to western blot analysis and GST pull down assay. To further address the role of R-Ras in transformation-related phenotype formation of breast cancer cell line MCF-7 in vitro, R-Ras38V, a constitutively activated mutant of R-Ras, was transfected into MCF-7 cells, and the cell proliferation, migration and cell cycle distribution were analyzed. The results showed that although there was slight difference in the protein expression of R-Ras between the breast cancer tissues and normal tissues, the activation of R-Ras was reduced in 63.8% of the cancer tissues when compared to the normal tissue samples. In addition, the results also showed that R-Ras38V inhibited cell proliferation, migration and cell cycle progression in the presence of serum. Contradicting the positive association reported in previous studies, our results indicate that R-Ras activation may negatively regulate the transformation of breast epithelial cells, and the loss of activation of R-Ras may be involved in the carcinogenesis of breast cancer. To solve this controversy, further independent studies are needed to validate our study results.