Characterizing genes associated with leukemic cell differentiation may provide help for understanding mechanisms on the leukemia differentiation. The aim of this study is to investigate whether the expression of melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) could be induced during leukemia differentiation and whether mda-7/IL-24 plays a role in leukemia differentiation. We showed that the expression of mda-7/IL-24 and IL-24 delE5, an mda-7/IL-24 splice variant, was induced in U937 and HL60 cells during 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated monocytic differentiation. Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway was required for their induction. Knockdown of mda-7/IL-24 and IL-24 delE5 resulted in significant inhibition of the monocytic differentiation induced by TPA. More importantly, ectopic overexpression of mda-7/IL-24 and IL-24 delE5 significantly induced U937 cells, HL60 cells, and blast cells from patients with acute myeloid leukemia-M5 to differentiate, whereas normal hematopoietic progenitors were not affected. Furthermore, the molecular effector associated with selective differentiation induction by mda-7/IL-24 and IL-24 delE5 may be reactive oxygen species (ROS), and the source of ROS generation was nicotinamide adenine dinucleotide phosphate oxidase. Taken together, our results reveal the mechanism by which TPA induces monocytic differentiation and show for the first time the specific differentiation-inducing effects of mda-7/IL-24 and IL-24 delE5 on human myeloid leukemic cells.