miRNAs play important roles in many biological processes, including erythropoiesis. Although several miRNAs regulate erythroid differentiation, how the key erythroid regulator, GATA-1, directly orchestrates differentiation through miRNA pathways remains unclear. In this study, we identified miR-23a as a key regulator of erythropoiesis, which was upregulated both during erythroid differentiation and in GATA-1 gain-of-function experiments, as determined by miRNA expression profile analysis. In primary human CD34+ hematopoietic progenitor cells, miR-23a increased in a GATA-1-dependent manner during erythroid differentiation. Gain- or loss-of-function analysis of miR-23a in mice or zebrafish demonstrated that it was essential for normal morphology in terminally differentiated erythroid cells. Furthermore, a protein tyrosine phosphatase, SHP2, was identified as a downstream target of miR-23a that mediated its regulation of erythropoiesis. Taken together, our data identify a key GATA-1-miRNA axis in erythroid differentiation.