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 alpha (ERalpha) is a substrate for protein kinases, and phosphorylation has profound effects on the function of this receptor. In this study, we show that ERalpha associates with c-Abl nonreceptor tyrosine kinase. The direct interaction is mediated by two PXXP motifs of ERalpha and the c-Abl SH3 domain. Mutational analysis and in vitro kinase assays show that ERalpha can be phosphorylated on two sites, tyrosine 52 (Y-52) and tyrosine 219 (Y-219). ERalpha phosphorylation by c-Abl stabilizes ERalpha, resulting in enhanced ERalpha transcriptional activity and increased expression of endogenous ERalpha target genes. Furthermore, ERalpha phosphorylation at the Y-219 site affects DNA binding and dimerization by ERalpha. Both the c-Abl inhibitor and the c-Abl kinase dead mutation abolish the c-Abl-induced accumulation of ERalpha and enhancement of ERalpha transcriptional activity, indicating that c-Abl kinase activity is required for regulation of the ERalpha function. Moreover, the ERalpha (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 ERalpha expression and promotes breast cancer cell proliferation, suggesting a great potential for this kinase to function as a therapeutic target for breast cancer.