Purpose: To assess the clinical significance of the interaction between estrogen and Runx2 signaling, previously shown in vitro. Experimental Design: MCF7/Rx2 dox breast cancer cells were treated with estradiol and/or doxycycline to induce Runx2, and global gene expression was profiled to define genes regulated by estradiol, Runx2, or both. Anchorage-independent growth was assessed by soft-agar colony formation assays. Expression of gene sets defined using the MCF7/Rx2 dox system was analyzed in pre- and on-treatment biopsies from hormone receptor-positive patients undergoing neoadjuvant letrozole treatment in two independent studies, and short-term changes in gene expression were correlated with tumor size reduction or Ki67 index at surgery. Results: Reflecting its oncogenic property, estradiol strongly promoted soft-agar colony formation, whereas Runx2 blocked this process suggesting tumor suppressor property. Transcriptome analysis of MCF7/Rx2 dox cells treated with estradiol and/or doxycycline showed reciprocal attenuation of Runx2 and estrogen signaling. Correspondingly in breast cancer tumors, expression of estradiol- and Runx2-regulated genes was inversely correlated, and letrozole increased expression of Runx2-stimulated genes, as defined in the MCF7/Rx2 dox model. Of particular interest was a gene set upregulated by estradiol and downregulated by Runx2 in vitro; its short-term response to letrozole treatment associated with tumor size reduction and Ki67 index at surgery better than other estradiol-regulated gene sets. Conclusion: This work provides clinical evidence for the importance of antagonism between Runx2 and E2 signaling in breast cancer. Likely sensing the tension between them, letrozole responsiveness of a genomic node, positively regulated by estradiol and negatively regulated by Runx2 in vitro, best correlated with the clinical efficacy of letrozole treatment.