Inhibition of protein kinase A (PKA) promotes estrogen-dependent growth of MCF7 breast cancer cells although the mechanisms by which PKA regulates estrogen receptor (ER) function remain unclear. In this study elevation of cAMP by Forskolin/IBMX (F/I) suppressed estradiol-dependent MCF7 and T47D breast cancer cell growth but not tamoxifen-resistant MCF7-LCC2 cells. Although F/I induced ligand independent activation of ER, F/I also decreased estradiol-dependent reporter gene transcription. Overexpression of PKA or PKA inhibitor (PKI) demonstrated that F/I effects on repression of estradiol action occurred through the PKA pathway. 8CPT-2Me-cAMP, a selective inducer of non-PKA signaling did not alter ER-dependent transcription. In contrast to F/I effects on reporter genes, F/I exhibited gene specific effects on endogenous, ER-regulated genes. F/I enhanced estradiol induction of pS2 and cMyc but repressed estradiol induction of cyclin D1 mRNA and protein in MCF7 cells. To explore likely mechanisms by which F/I regulated ER, experiments examined estradiol binding, Hsp90 interaction, promoter recruitment and ER phosphorylation. F/I decreased estradiol binding and increased Hsp90 association with ER. Chromatin immunoprecipitation revealed that F/I recruited ER to both pS2 and cMyc promoters at earlier times than estradiol and F/I shifted estradiol recruitment of ER to earlier time points. F/I induced a unique ER phosphorylation profile (increase in serine 305 and decrease in serine 118 phosphorylation) that was distinct from estradiol and estradiol+F/I. Taken together, F/I signaling through PKA selectively regulates estradiol-dependent genes in breast cancer that is associated with reduced ligand binding and changes in promoter interaction and ER phosphorylation.