Alteration of protein O-glycosylation in various human cancers including breast cancer is well known， but molecular roles of their aberrant glycosylations on cancer have not been fully understood. We previously reported critical roles of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6 or GalNAc-T6) that was upregulated in a great majority of breast cancer tissues. Here we further report O-glycosylation of estrogen receptor alpha (ER-α) by GALNT6 and the significant role of its nuclear localization in breast cancer cells. Knockdown of GALNT6 expression in two breast cancer cell lines， T47D and MCF7， in which both ER-α and GALNT6 were highly expressed， by small interfering RNA could significantly attenuate expression of ER-α. Immunocytochemical analysis clearly demonstrated the drastic decrease of ER-α protein in the nucleus of these cancer cells. Accordingly， the downstream genes of the ER-α pathway such as MYC， CCND1， and CTSD were significantly downregulated. We confirmed GALNT6-dependent ER-α O-glycosylation and identified O-glycosylation of S573 in an F domain of ER-α by GALNT6 through LC-MS/MS analysis. We also obtained evidences showing that the glycosylation of ER-α at S573 by GALNT6 is essential for protein stability and nuclear localization of ER-α in breast cancer cells. Furthermore， we designed cell membrane-permeable peptides including the O-glycosylation site and found a significant decrease of the cell viability of breast cancer cells by treatment of these peptides in a GALNT6 expression-dependent manner. Our study suggests that targeting the GALNT6 enzymatic activity as well as the GALNT6/ER-α interaction could be a promising therapeutic approach to ER-α-positive breast cancer patients.