Our study shows for the first time that melatonin inhibits proliferation and induces apoptosis in FLT3/ITD-positive leukemia cells. Mechanistically, melatonin preferentially causes overproduction of reactive oxygen species (ROS) and ultimately massive cell death in FLT3-ITD AML cells. Moreover, melatonin significantly enhances the cytotoxicity induced by the FLT3 tyrosine kinase inhibitor sorafenib in AML cells with FLT3/ITD through redox modification. Importantly, combination of melatonin and sorafenib exhibited highly synergistic therapeutic activity in MV4-11 xenografts and a murine model bearing FLT3/ITD leukemia.