This study aims to investigate the clinical significance and biological function of RASSF6 in human breast cancers. RASSF6 protein was found to be downregulated in 42 of 95 human breast cancer tissues by immunohistochemistry, which was associated with advanced TNM stage and nodal metastasis. The rate of RASSF6 downregulation was higher in Triple-negative breast cancer (TNBC). Downregulation of RASSF6 protein was also found in breast cancer cell lines, especially in TNBC cell lines. Overexpression RASSF6 inhibited cell growth rate and colony formation ability in MDA-MB-231 cell line. Depletion of RASSF6 promoted proliferation rate and colony formation ability in T47D cell line. Flow cytometry/PI staining demonstrated that RASSF6 inhibited cell cycle transition. AnnxinV/PI analysis showed that RASSF6 overexpression upregulated apoptosis induced by cisplatin (CDDP) while RASSF6 depletion inhibited apoptosis. JC-1 staining showed that RASSF6 overexpression inhibited mitochondrial membrane potential. Western blot analysis demonstrated that RASSF6 repressed cyclin D1, YAP while upregulated p21, cleaved caspase 3 and cytochrome c expression. In addition, RASSF6 activated Hippo signaling pathway by upregulating MST1/2 and LATS1 phosphorylation. Restoration of YAP inhibited cleaved caspase 3 and cytochrome c which were induced by RASSF6. Restoration of YAP also reduced the rate of CDDP induced apoptosis. In conclusion, this study provided evidence that RASSF6 functions as a potential tumor suppressor in human breast cancer through activation of Hippo pathway. (C) 2018 Elsevier Inc. All rights reserved.