ER (estrogen receptor)-α36, a variant of human ERα, activates non-genomic cell signaling pathways. ER-α36 on the cell membrane plays a role in breast cancer growth and development, and contributes to tamoxifen resistance. However, it is not understood how cell membrane expression of ER-α36 is regulated. In this study, we investigated the role of cell membrane glycoprotein 96 (mgp96) in the regulation of ER-α36 expression and signaling. We found that the C-terminal domain of mgp96 directly interacts with ER-α36 on the cell membrane of breast tumor cells. This interaction stabilizes the ER-α36 protein, thereby increasing its signaling, which, in turn, increases tumor cell growth and invasion. Moreover, targeting mgp96 with siRNA or monoclonal antibody (mAb) blocks the mgp96-ER-α36 interaction and inhibits breast cancer growth and invasion both in vitro and in vivo. These results provide insights into the modulation of cell membrane ER-α36 expression and suggest that mgp96 could be a potential therapeutic target for ER-α36-overexpressing breast cancer.