microRNAs (miRNAs) are involved in a variety of biological processes. The regulatory function and potential role of miRNAs targeting the mRNA of the 5-aminolevulinate synthase 2 (ALAS2) in erythropoiesis were investigated in order to identify miRNAs which play a role in erythroid iron metabolism and differentiation. Firstly, the role of ALAS2 in erythroid differentiation and iron metabolism in human erythroid leukemia cells (K562) was confirmed by ALAS2 knockdown. Through a series of screening strategies and experimental validations, it was identified that hsa-miR-218 (miR-218) targets and represses the expression of ALAS2 by binding to the 3-untranslated region (UTR). Overexpression of miR-218 repressed erythroid differentiation and altered iron metabolism in K562 cells similar to that seen in the ALAS2 knockdown in K562 cells. In addition to iron metabolism and erythroid differentiation, miR-218 was found to be responsible for a reduction in K562 cell growth. Taken together, our results show that miR-218 inhibits erythroid differentiation and alters iron metabolism by targeting ALAS2 in K562 cells.