Background: Metastasis remains the biggest obstacle for breast cancer treatment. Therefore, identification of specific biomarker of metastasis is very necessary. The RNA binding protein 3 (RBMS3) acts as a tumor suppressor in various cancers. Whereas, its role and underlying molecular mechanism in breast cancer is far from elucidated. Methods: Quantitative real-time PCR and western blots were carried out to determine the expression of RBMS3 in breast cancer cells and tissues. Transwell and in vivo metastasis assay were conducted to investigate the effects of RBMS3 on migration, invasion and metastasis of breast cancer cells. Transcriptome sequencing was applied to screen out the differential gene expression affected by RBMS3. RNA immunoprecipitation assay combined with luciferase reporter assay were performed to explore the direct correlation between RBMS3 and Twist1 mRNA. Results: RBMS3 was downregulated in breast cancer and ectopic expression of RBMS3 contributed to inhibition of cell migration, invasion in vitro and lung metastasis in vivo. Furthermore, RBMS3 negatively regulated Twsit1 expression via directly binding to 3′-UTR of Twist1 mRNA, and thereby decreased Twist1-induced expression of matrix metalloproteinase 2 (MMP2). Additionally, Twist1-induced cell migration, invasion and lung metastasis could be reversed by the upregulation of RBMS3. Conclusions: In summary, our study revealed a novel mechanism of the RBMS3/Twsit1/MMP2 axis in the regulation of invasion and metastasis of breast cancer, which may become a potential molecular marker for breast cancer treatment.