MiR-424 plays an important role via promoting the monocytic differentiation in many human leukemia cell lines. Here, we report that miR-424 decreased miR-125b expression to 36 % by directly targeting caudal type homeobox 2. However, miR-424 also decreased expression of Fes, PU.1 and colony-stimulating factor receptor (MCSFR). As Fes, PU.1 and MCSFR were down-regulated by over-expression of miR-125b (unpublished work), a similar effect of miR-424 and Fes siRNA on CD64, Egr-1, Egr-2 and CEBPA indicates that Fes may be an important downstream target of miR-424. We hypothesize that miR-424 promotes monocytic differentiation by regulating other critical factors and miR-424 has high affinity for these factors. For the first time, the molecular mechanism of miR-424 during monocytic differentiation of U937 cells has been elucidated in this study.