Human myeloid leukemia cells exposed to 1,25-dihydroxyvitamin D(3) (1,25D), a major cancer chemopreventive agent, acquire features of normal monocytes and arrest in the G(1) phase of the cell cycle, due to the upregulation of p27(Kip1) and p21(Cip1), but the mechanism is not clear. Here evidence is provided that an exposure of HL60 and U937 cells to low (1-10 nM) concentrations of 1,25D decreases the expression of miR181a and miR181b in a concentration and time-dependent manner. Since the predicted miR181 targets include the 3'-UTR of p27(Kip1), we expressed pre-miR181a in these cells, and found that the elevation of cellular miR181a levels abrogates the 1,25D-induced increase in p27(Kip1) at both mRNA and protein levels. In contrast, transfection of pre-miR181a resulted in a slight elevation of p21(Cip1) expression. Importantly, transfection of pre-miR181a blunted the effect of 1,25D on the expression of monocytic differentiation markers, and reduced the G(1) block in 1,25D-treated cells, while transfection of anti-miR181a increased 1,25D-induced differentiation. Together, these data show that miR181a participates in 1,25D-induced differentiation of HL60 and U937 cells, and suggest that a high constitutive expression of members of miR181 family may contribute to the malignant phenotype in the myeloid lineage.