We previously demonstrated globin gene inhibition in K562 cells and primary erythroid progenitors treated with Interleukin-6. Although several cis-acting elements have been identified in the globin promoters, the precise mechanism for cytokine-mediated globin gene regulation remains to be elucidated. In this report we demonstrate inhibitors of Stat3 phosphorylation abrogate Interleukin-6-mediated gene silencing in erythroid cells. DNA-protein binding studies established Stat3 interaction in the 5'-untranslated globin promoter region. Furthermore, co-transfection experiments with Stat3 demonstrate promoter inhibition in a concentration-dependent manner, which was significantly reversed when the cognate Stat3 binding site in the 5'-untranslated region was mutated. These studies establish a novel mechanism for globin gene silencing through the STAT signal transduction pathway.