Polyamines are required for maintenance of intestinal epithelial integrity, and a decrease in cellular polyamines increases the cytoplasmic levels of RNA-binding protein HuR stabilizing p53 and nucleophosmin mRNAs, thus inhibiting IEC (intestinal epithelial cell) proliferation. The AMPK (AMP-activated protein kinase), an enzyme involved in responding to metabolic stress, was recently found to be implicated in regulating the nuclear import of HuR. Here, we provide evidence showing that polyamines modulate subcellular localization of HuR through AMPK-regulated phosphorylation and acetylation of Impα1 (importin α1) in IECs. Decreased levels of cellular polyamines as a result of inhibiting ODC (ornithine decarboxylase) with DFMO (D,L-α-difluoromethylornithine) repressed AMPK activity and reduced Impα1 levels, whereas increased levels of polyamines as a result of ODC overexpression induced both AMPK and Impα1 levels. AMPK activation by overexpression of the AMPK gene increased Impα1 but reduced the cytoplasmic levels of HuR in control and polyamine-deficient cells. IECs overexpressing wild-type Impα1 exhibited a decrease in cytoplasmic HuR abundance, while cells overexpressing Impα1 proteins bearing K22R (lacking acetylation site), S105A (lacking phosphorylation site) or K22R/S105A (lacking both sites) mutations displayed increased levels of cytoplasmic HuR. Ectopic expression of these Impα1 mutants also prevented the increased levels of cytoplasmic HuR following polyamine depletion. These results indicate that polyamine-mediated AMPK activation triggers HuR nuclear import through phosphorylation and acetylation of Impα1 in IECs and that polyamine depletion increases cytoplasmic levels of HuR as a result of inactivation of the AMPK-driven Impα1 pathway.