AbstractChromobox 6 (CBX6) is a subunit of Polycomb Repressive Complex 1 (PRC1) that mediates epigenetic gene repression and acts as an oncogene or tumor suppressor in a cancer type-dependent manner. The specific function of CBX6 in breast cancer is currently undefined. In this study, a comprehensive analysis of The Cancer Genome Atlas (TCGA) dataset led to the identification of CBX6 as a consistently downregulated gene in breast cancer. We provided evidence showing enhancer of zeste homolog 2 (EZH2) negatively regulated CBX6 expression in a Polycomb Repressive Complex 2 (PRC2)-dependent manner. Exogenous overexpression of CBX6 inhibited cell proliferation and colony formation, and induced cell cycle arrest along with suppression of migration and invasion of breast cancer cells in vitro. Microarray analyses revealed that CBX6 governs a complex gene expression program. Moreover, CBX6 induced significant downregulation of bone marrow stromal cell antigen-2 (BST2), a potential therapeutic target, via interactions with its promoter region. Our collective findings support a tumor suppressor role of CBX6 in breast cancer.