Estrogen receptors are members of the steroid hormone superfamily of nuclear receptors that act as ligand-activated transcription factors. Similar to other steroid hormone receptors, estrogen receptor α (ERα) is a substrate for protein kinases, and phosphorylation has profound effects on the function of this receptor. In this study, we show that ERα associates with c-Abl nonreceptor tyrosine kinase. The direct interaction is mediated by two PXXP motifs of ERα and the c-Abl SH3 domain. Mutational analysis and in vitro kinase assays show that ERα can be phosphorylated on two sites, tyrosine 52 (Y-52) and tyrosine 219 (Y-219). ERα phosphorylation by c-Abl stabilizes ERα, resulting in enhanced ERα transcriptional activity and increased expression of endogenous ERα target genes. Furthermore, ERα phosphorylation at the Y-219 site affects DNA binding and dimerization by ERα. Both the c-Abl inhibitor and the c-Abl kinase dead mutation abolish the c-Abl-induced accumulation of ERα and enhancement of ERα transcriptional activity, indicating that c-Abl kinase activity is required for regulation of the ERα function. Moreover, the ERα (Y52, 219F) mutant shows reduced breast cancer cell growth and invasion. Taken together, these results show that c-Abl is a novel kinase that upregulates ERα expression and promotes breast cancer cell proliferation, suggesting a great potential for this kinase to function as a therapeutic target for breast cancer.