The incidence of breast cancer is increasing throughout the world. Although significant progress has been made in diagnostic techniques and targeted therapies, the prognosis of breast cancer remains poor. Regulator of differentiation 1 (ROD1) may inhibit the development of several types of cancer. However, the role of ROD1 in breast cancer cells remains unknown. In the present study, western blot analysis and reverse transcription-quantitative polymerase chain reaction revealed that expression of ROD1 was significantly reduced in breast cancer cells. Overexpression of ROD1 reduced the proliferation rate, demonstrated using a Cell Counting Kit-8 assay. Additionally, the overexpression of ROD1 decreased the invasiveness of breast cancer cells, indicating that ROD1 may serve as a tumor suppressor. Additionally, the data suggested that ROD1 significantly suppressed the activity of Wnt luciferase reporter (TOP Flash) in MDA-MB-231 cells. Furthermore, it was demonstrated that ROD1 may interact with beta-catenin by using co-immunoprecipitation, resulting in suppression of beta-catenin migration into the nucleus. Notably, ROD1 demonstrated its anticancer effect by decreasing beta-catenin (Y333) phosphorylation in a nude mouse xenograft model. Overexpression of ROD1 may downregulate Ki67 protein levels, as determined by immunohistochemistry. These results indicated that ROD1 may be used as a therapeutic target in patients with breast cancer.