Abstract Purpose: Determine the roles of the PI3K isoforms p110α and p110β in PTEN-deficient, estrogen receptor α (ER)-positive breast cancer, and the therapeutic potential of isoform-selective inhibitors. Experimental Design: Anti-estrogen-sensitive and -resistant PTEN-deficient, ER+ human breast cancer cell lines, and mice bearing anti-estrogen–resistant xenografts were treated with the anti-estrogen fulvestrant, the p110α inhibitor BYL719, the p110β inhibitor GSK2636771, or combinations. Temporal response to growth factor receptor–initiated signaling, growth, apoptosis, predictive biomarkers, and tumor volumes were measured. Results: p110β primed cells for response to growth factor stimulation. Although p110β inhibition suppressed cell and tumor growth, dual targeting of p110α/β enhanced apoptosis and provided sustained tumor response. The growth of anti-estrogen–sensitive cells was inhibited by fulvestrant, but fulvestrant inconsistently provided additional therapeutic effects beyond PI3K inhibition alone. Treatment-induced decreases in phosphorylation of AKT and Rb were predictive of therapeutic response. Short-term drug treatment induced tumor cell apoptosis and proliferative arrest to induce tumor regression, whereas long-term treatment only suppressed proliferation to provide durable regression. Conclusions: p110β is the dominant PI3K isoform in PTEN-deficient, ER+ breast cancer cells. Upon p110β inhibition, p110α did not induce significant reactivation of AKT, but combined targeting of p110α/β most effectively induced apoptosis in vitro and in vivo and provided durable tumor regression. Because apoptosis and tumor regression occurred early but not late in the treatment course, and proliferative arrest was maintained throughout treatment, p110α/β inhibitors may be considered short-term cytotoxic agents and long-term cytostatic agents. Clin Cancer Res; 23(11); 2795–805. ©2016 AACR.