MicroRNAs, a class of small noncoding RNAs, have been implicated to regulate gene expression in virtually all important biological processes. Although accumulating evidence demonstrates that miR-150, an important regulator in hematopoiesis, is deregulated in various types of hematopoietic malignancies, the precise mechanisms of miR-150 action are largely unknown. In this study, we found that miR-150 is downregulated in samples from patients with acute lymphoblastic leukemia, acute myeloid leukemia, and chronic myeloid leukemia, and normalized after patients achieved complete remission. Restoration of miR-150 markedly inhibited growth and induced apoptosis of leukemia cells, and reduced tumorigenicity in a xenograft leukemia murine model. Microarray analysis identified multiple novel targets of miR-150, which were validated by quantitative real-time PCR and luciferase reporter assay. Gene ontology and pathway analysis illustrated potential roles of these targets in small-molecule metabolism, transcriptional regulation, RNA metabolism, proteoglycan synthesis in cancer, mTOR signaling pathway, or Wnt signaling pathway. Interestingly, knockdown one of four miR-150 targets (EIF4B, FOXO4B, PRKCA, and TET3) showed an antileukemia activity similar to that of miR-150 restoration. Collectively, our study demonstrates that miR-150 functions as a tumor suppressor through multiple mechanisms in human leukemia and provides a rationale for utilizing miR-150 as a novel therapeutic agent for leukemia treatment.