Objective(s): A growing body of evidence indicates that rhomboid domain containing 1 (RHBDD1) plays an important role in a variety of physiological and pathological processes, including tumorigenesis. We aimed to determine the function of RHBDD1 in breast cancer cells. Materials and Methods: In this study, we used the Oncomine™ database to determine the expression patterns of RHBDD1 in normal and breast cancer tissues. We performed lentiviral transfection of RHBDD1-specific small interfering RNA into the breast cancer cell lines ZR-75-30 and MDA-MB-231 in order to investigate the effects of RHBDD1 deficiency on breast cancer metastasis. Results: We found that knockdown of RHBDD1 inhibited breast cancer cell migration and invasion in vitro. Moreover, knockdown of RHBDD1 promoted epithelial–mesenchymal transition (EMT) by suppressing the expression of MPP2, MPP9, fibronectin 1, vimentin, SRY-box 2, zinc finger E-box binding homeobox 1, and snail family transcriptional repressor 1, and promoting the expression of cadherin 1. Additionally, knockdown of RHBDD1 inhibited the protein expression and phosphorylation of Akt.Conclusion: Our data indicate that RHBDD1 overexpression may promote breast cancer metastasis via the regulation of EMT, suggesting that RHBDD1 may be an important regulator of breast cancer metastasis