Abstract To understand how microRNAs (miRNAs) regulate breast cancer tumorigenesis, a miRNA expression microarray screening was performed using RNAs from formalin-fixed paraffin-embedded (FFPE) breast tissues, which included benign (n=13), ductal carcinoma in situ (DCIS) (n=16), and invasive ductal carcinoma (IDC) (n=15). Twenty miRNAs that were differentially expressed (p<0.01) were identified, of which let-7 family miRNAs were down-regulated in human breast cancer tissues at stages of DCIS and IDC compared to benign stage. To understand the role of let-7 miRNAs further, we performed bioinformatics analysis and found that let-7 miRNA sequences match sequences in the 3’-UTRs of estrogen receptor alpha 66 (ER-α66) and another novel receptor ER-α36. The targeting of let-7 miRNAs on ER-α66 and ER-α36 was further confirmed by a number of experimental assays, including luciferase assay, protein expression, and mRNA expression. Overexpression of let-7 miRNAs in ER-α66-positive breast cancer MCF7 cell line negatively affected ER-α66 mediated genomic estrogen pathway. As expected, down-regulation of the ER-α66 signaling by let-7 miRNAs inhibited cell proliferation, and subsequently triggered the cell apoptotic process in MCF7 cells. On the other hand, overexpression of let-7 miRNAs in ER-α66-negative cell line MB-MDA-231 negatively affected ER-α36 mediated non-genomic estrogen pathway. Besides, down-regulation of let-7 is correlated with up-regulation of ER-α36 in tamoxifen (Tam) and ICI-182780 (ICI) resistance in MCF7 cell line. Forced overexpression in Tam-and ICI-resistant MCF7 cells dampened ER-α36 expression. In conclusion, our findings not only indicate a new regulatory mechanism of let-7 miRNAs in ER mediated cellular malignant growth of breast cancer, and but also provide potential biomarkers and/or surrogate therapeutic targets useful for early diagnosis and/or therapeutic options for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2025.