Predicting the impact of microRNAs (miRNAs) on target proteins is challenging because of their different regulatory effects at the transcriptional and translational levels. In this study, we applied a novel protein lysate microarray (LMA) technology to systematically monitor for target protein levels after high-throughput transfections of 319 pre-miRs into breast cancer cells. We identified 21 miRNAs that downregulated the estrogen receptor-α (ERα), as validated by western blotting and quantitative real time–PCR, and by demonstrating the inhibition of estrogen-stimulated cell growth. Five potent ERα-regulating miRNAs, miR-18a, miR-18b, miR-193b, miR-206 and miR-302c, were confirmed to directly target ERα in 3′-untranslated region reporter assays. The gene expression signature that they repressed highly overlapped with that of a small interfering RNA against ERα, and across all the signatures tested, was most closely associated with the repression of known estrogen-induced genes. Furthermore, miR-18a and miR-18b showed higher levels of expression in ERα-negative as compared with ERα-positive clinical tumors. In summary, we present systematic and direct functional evidence of miRNAs inhibiting ERα signaling in breast cancer, and demonstrate the high-throughput LMA technology as a novel, powerful technique in determining the relative impact of various miRNAs on key target proteins and associated cellular processes and pathways.