Pancreatic cancer is one of the most highly fatal cancers and is generally resistant to chemotherapy. Currently, gemcitabine appears to be the only effective agent for its treatment and is the preferred first-line therapy. However, the clinical impact of gemcitabine remains modest due to a high level of inherent and acquired tumor resistance. We investigated protein expression in gemcitabine-resistant and -sensitive human pancreatic adenocarcinoma cell lines by proteomics. Tumor cell proteins were separated by two-dimensional gel electrophoresis, then the protein spots that showed increased or decreased expression in gemcitabine-sensitive cell lines were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and Western blotting. Nine out of ten proteins showing differential expression in gemcitabine-resistant and -sensitive cell lines were identified, confirming an increase in heat shock protein 27 (HSP27) and a decrease in nucleophosmin (NPM) in the resistant lines. These results suggest that HSP27 and NPM may play a role in the poor response of pancreatic cancer to gemcitabine.