Differentiated macrophages are essential for the innate immune system; however, the molecular mechanisms underlying the generation of macrophages remain largely unknown. Here we show that the RNA-binding protein QKI, mainly QKI-5, is transcriptionally activated in the early differentiated monocytic progenitors when CCAAT/enhancer-binding protein (C/EBP) α is expressed. The forced expression of C/EBPα increases the endogenous expression of QKI. Chromatin immunoprecipitation analysis and reporter assays further confirm that C/EBPα activates the transcription of QKI, primarily by binding to the distal C/EBPα-binding site. Blocking the induction of QKI using RNA interference enhances the expression of endogenous CSF1R and facilitates macrophage differentiation. Further study of the mechanism reveals that QKI-5 facilitates the degradation of CSF1R mRNA by interacting with the distal QRE in the 3' untranslated region. In summary, we show that in committed macrophage progenitors, C/EBPα-activated QKI-5 negatively regulates macrophage differentiation by down-regulating CSF1R expression, forming a negative feedback loop during macrophage differentiation.