miR-15a and miR-16-1 (miR-15a/16-1) have been implicated in apoptosis, cell cycle regulation and chemosensitivity of tumor cells, but little is known about the role of miR-15a/16-1 in the differentiation of leukemic cells. In this study, we found that the expression level of miR-15a/16-1 was up-regulated by all-trans retinoic acid (ATRA) treatment in NB4, HL-60 and U937 cell lines and primary leukemic cells. Overexpression of miR-15a/16-1 could not directly drive cells to undergo differentiation but enhanced ATRA-induced differentiation in NB4 and U937 cells. Up-regulation of miR-15a/16-1 was also observed in 36 patients with acute myeloid leukemia (AML) who achieved a complete remission (CR), and two of them showed sharp down-regulation of miR-15a/16-1 when they had a molecular relapse. These data indicate that miR-15a/16-1 plays an important role in the ATRA-induced differentiation of leukemic and primary AML cells.