Peroxiredoxin III (Prdx III), the mitochondrial peroxidase, was preferentially expressed in murine erythroleukemia (MEL) cells. However, the mechanisms by which Prdx III regulates erythroid differentiation are unknown. In this study, K562 cells were differentiated by Ara-C treatment, and Prdx III was dramatically increased until day 5. We also investigated Prdx III expression pattern on in vitro erythropoiesis of human CD34(+) cells. When human CD34(+) cells became proerythrocyte on day 7, Prdx III was diminished, and then augmented on day 12. We established the stable sublines of Prdx III overexpression (O/E), and dominant-negative (D/N). The intracellular ROS level of Prdx III O/E cell line was lower than D/N stable cell lines. Moreover, Prdx III O/E cell line was placed in G1-arrest, but not D/N cell lines. Finally, the expression level of beta-globin and GATA-1 was dramatically increased in Prdx III O/E cell line.