Estrogen receptor-alpha (ERalpha) and its ligand estradiol (E2) has critical roles in breast cancer growth and are key therapeutic targets. In this study, we report a novel dual role of the adenosine A1 receptor (Adora1) as an E2/ERalpha target and a regulator of ERalpha transcriptional activity. In ERalpha-positive breast cancer cells, E2 upregulated Adora1 messenger RNA (mRNA) and protein levels, an effect that was reversed by the E2 antagonist ICI 182 780. Small interference RNA ablation of Adora1 in ERalpha-positive cells reduced basal and E2-dependent proliferation, whereas Adora1 over-expression in an ERalpha-negative cell line induced proliferation. The selective Adora1 antagonist, DPCPX, reduced proliferation, establishing Adora1 as a mediator of E2/ERalpha-dependent breast cancer growth. Intriguingly, Adora1 ablation decreased both mRNA and protein levels of ERalpha and, consequently, estrogen-responsive element-dependent ERalpha transcriptional activity. Moreover, Adora1 ablation decreased binding activity of ERalpha to the promoter of its target gene TFF1 and led to reduced TFF1 promoter activity and mRNA levels, suggesting that Adora1 is required for full transcriptional activity of ERalpha on E2 stimulation. Taken together, we showed a short feed-forward loop involving E2, ERalpha and Adora1 that favors breast cancer growth. These data suggest that Adora1 may represent an important target for therapeutic intervention in hormone-dependent breast cancer.