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 α (ERα) mRNA and protein. RNA interference studies confirmed that basal expression of ERα was dependent on Sp1 but not Sp3 or Sp4 in MCF-7 cells. as-miR-27a and overexpression of ZBTB10 inhibited 17β-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 G₀/G₁ and inhibited E2-induced G₀/G₁ 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 G₂/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 ERα.