Micro RNAs are a class of small non-coding RNAs which has been recently shown to play a crucial role in major cellular processes such as development and differentiation through post-transcriptional regulation. The role of these epigenetic elements has also been demonstrated in hematopoietic lineage differentiation and there is a large body of evidence that miR-424 is responsible for monocyte differentiation. Our goal was to examine the effect of miR-424 over-expression on defeating the maturation blockage in monoblastic cell line U937. The permanent over-expression of miR-424 was established using a retroviral vector construct containing the precursor of miR-424 sequence. Induction of differentiation process was monitored by assaying changes in cell morphology, and expression of cell surface markers using light microscopy, quantitative RT-PCR, and flow cytometry for monocyte markers such as CD11b and CD14. The cells showed monocytic characteristics 14 days after transduction, and CD11b and CD14 expression were significantly increased, confirmed by flow cytometry QRT-PCR and RT-PCR results. In conclusion, miR-424 over-expression is an effective factor in maturation of the monoblastic U937 cells and it has the ability of directing them into cells, expressing monocyte/macrophage characteristics.