Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, whereas acute myeloid leukemia (AML) is the most common acute leukemia in adults. In general, ALL has a better prognosis than AML. To understand the distinct mechanisms in leukemogenesis between ALL and AML and to identify markers for diagnosis and treatment, we performed a large-scale genome-wide microRNA (miRNA, miR) expression profiling assay and identified 27 miRNAs that are differentially expressed between ALL and AML. Among them, miR-128a and -128b are significantly overexpressed, whereas let-7b and miR-223 are significantly down-regulated in ALL compared with AML. They are the most discriminatory miRNAs between ALL and AML. Using the expression signatures of a minimum of two of these miRNAs resulted in an accuracy rate of >95% in the diagnosis of ALL and AML. The differential expression patterns of these four miRNAs were validated further through large-scale real-time PCR on 98 acute leukemia samples covering most of the common cytogenetic subtypes, along with 10 normal control samples. Furthermore, we found that overexpression of miR-128 in ALL was at least partly associated with promoter hypomethylation and not with an amplification of its genomic locus. Taken together, we showed that expression signatures of as few as two miRNAs could accurately discriminate ALL from AML, and that epigenetic regulation might play an important role in the regulation of expression of miRNAs in acute leukemias.