Using a novel approach that detects changes in the conformation of ERα, we studied the efficacy of anti-estrogens to inactivate ERα under different experimental conditions. We show that phosphorylation of serine-305 in the hinge region of ERα by protein kinase A (PKA) induced resistance to tamoxifen. Tamoxifen bound but then failed to induce the inactive conformation, invoking ERα-dependent transactivation instead. PKA activity thus induces a switch from antagonistic to agonistic effects of tamoxifen on ERα. In clinical samples, we found that downregulation of a negative regulator of PKA, PKA-RIα, was associated with tamoxifen resistance prior to treatment. Activation of PKA by downregulation of PKA-RIα converts tamoxifen from an ERα inhibitor into a growth stimulator, without any effect on ICI 182780 (Fulvestrant).