The role of microRNA in the aberrant autophagy that occurs in pancreatic cancer remains controversial. Because hypoxia is known to induce autophagy, we screened for differentially expressed microRNAs using a miRNA microarray with pancreatic cancer cells cultured under normoxic and hypoxic conditions. We found that miR-138-5p was among the most downregulated miRNA in hypoxia-stimulated cells, and that overexpression of miR-138-5p substantially reduced expression of autophagy markers. In addition, western blot and immunofluorescence analyses and electron microscopy revealed that miR-138-5p inhibited autophagy in pancreatic cancer cells and blocked serum starvation-induced autophagic flux independently of the typical autophagic signaling pathway. miR-138-5p had no effect on ATG3, ATG5, or ATG7, three primary autophagy-associated genes. Instead, miR-138-5p specifically targeted the SIRT1 3' untranslated region and suppressed autophagy by reducing the level of SIRT1, which acetylates FoxO1 and regulates autophagy via FoxO1/Rab7. SIRT1 or Rab7 knockdown blocked the SIRT1/FoxO1/Rab7 axis and suppressed autophagic inhibition by miR-138-5p. Finally, we found that miR-138-5p inhibited autophagy and tumor growth in vivo. These results indicate that miR-138-5p suppresses autophagy in pancreatic cancer by targeting SIRT1.