All mammalian cells depend on polyamines for normal growth and proliferation, but the exact roles of polyamines at the molecular level remain largely unknown. The RNA-binding protein HuR modulates the stability and translation of many target mRNAs. Here, we show that in rat intestinal epithelial cells (IECs), polyamines enhanced HuR association with the 3′-untranslated region of the c-Myc mRNA by increasing HuR phosphorylation by Chk2, in turn promoting c-Myc translation. Depletion of cellular polyamines inhibited Chk2 and reduced the affinity of HuR for c-Myc mRNA; these effects were completely reversed by addition of the polyamine putrescine or by Chk2 overexpression. In cells with high content of cellular polyamines, HuR silencing or Chk2 silencing reduced c-Myc translation and c-Myc expression levels. Our findings demonstrate that polyamines regulate c-Myc translation in IECs through HuR phosphorylation by Chk2 and provide new insight into the molecular functions of cellular polyamines.