Estrogen drives both transcriptional activation and proteolysis of estrogen receptor α (ERα; encoded by ESR1). Here we observed variable and overlapping ESR1 mRNA levels in 200 ERα-negative and 50 ERα-positive primary breast cancers examined, which suggests important posttranscriptional ERα regulation. Our results indicate that Src cooperates with estrogen to activate ERα proteolysis. Inducible Src stimulated ligand-activated ERα transcriptional activity and reduced ERα t 1/2. Src and ERα levels were inversely correlated in primary breast cancers. ERα-negative primary breast cancers and cell lines showed increased Src levels and/or activity compared with ERα-positive cancers and cells. ERα t 1/2 was reduced in ERα-negative cell lines. In both ERα-positive and-negative cell lines, both proteasome and Src inhibitors increased ERα levels. Src inhibition impaired ligand-activated ERα ubiquitylation and increased ERα levels. Src siRNA impaired ligand-activated ERα loss in BT-20 cells. Pretreatment with Src increased ERα ubiquitylation and degradation in vitro. These findings provide what we believe to be a novel link between Src activation and ERα proteolysis and support a model whereby crosstalk between liganded ERα and Src drives ERα transcriptional activity and targets ERα for ubiquitin-dependent proteolysis. Oncogenic Src activation may promote not only proliferation, but also estrogen-activated ERα loss in a subset of ERα-negative breast cancers, altering prognosis and response to therapy.