MicroRNA-27a (miR-27a) is expressed in MCF-7 breast cancer cells, and antisense miR-27a (as-miR-27a) induces ZBTB10, a specificity protein (Sp) repressor. Both as-miR-27a and overexpression of ZBTB10 decreased Sp1, Sp3, and Sp4 mRNA and protein expression in MCF-7 cells, and this was also accompanied by decreased levels of estrogen receptor alpha (ERalpha) mRNA and protein. RNA interference studies confirmed that basal expression of ERalpha was dependent on Sp1 but not Sp3 or Sp4 in MCF-7 cells. as-miR-27a and overexpression of ZBTB10 inhibited 17beta-estradiol (E2)-induced transactivation in MCF-7 cells, and this was accompanied by decreased binding of Sp and ER proteins in cell lysates to oligonucleotides containing GC-rich motifs or estrogen-responsive elements, respectively. as-miR-27a and overexpression of ZBTB10 arrested MCF-7 cells in G(0)/G(1) and inhibited E2-induced G(0)/G(1) to S phase progression. as-miR-27a induced only a minimal increase in Myt-1, another miR-27a regulated gene, and this was not accompanied by Myt-1-dependent G(2)/M arrest as observed previously in ER-negative MDA-MB-231 breast cancer cells. Thus, miR-27a indirectly regulates E2-responsiveness in MCF-7 cells through suppression of ZBTB10, thereby enhancing expression of ERalpha.