The role of the proteolytic enzyme elastase in motility and proliferation of leukemic human acute myeloblastic leukemia (AML) cells is currently unknown. We report a correlation between abnormally high levels of elastase in the blood of AML patients and the number of leukemic blast cells in the circulation. In AML cells, we observed expression of cell-surface elastase, which was regulated by the chemokine stromal cell-derived factor-1 (SDF-1). In vitro inhibition of elastase prevented SDF-1-induced cell polarization, podia formation, and reduced migration of human AML cells as well as their adhesion. Elastase inhibition also significantly impaired in vivo homing of most human AML cells to the bone marrow (BM) of nonobese diabetic-severe combined immunodeficient (NOD/SCID)/beta-2 microglobulin knock-out (B2m null) mice that underwent transplantation. Moreover, in vitro proliferation of AML cells was elastase dependent. In contrast, treatment with elastase inhibitor enhanced the proliferation rate of human cord blood CD34+ cells, including primitive CD34+/CD38- cells, and their in vivo homing. Finally, NOD/SCID mice previously engrafted with human AML cells and treated with elastase inhibitor had significantly reduced egress of leukemic cells into the circulation. Taken together, our data demonstrate that human AML cells constitutively secrete and express SDF-1-dependent cell-surface elastase, which regulates their migration and proliferation.