The prognosis of patients with pancreatic cancer is very poor because of late diagnosis and the lack of response to various therapies. Pancreatic cancer is generally resistant to chemotherapy and is highly fatal. Gemcitabine (GEM) appears to be the only effective agent for treatment of pancreatic cancer. However, a high level of inherent and acquired tumor resistance makes the clinical impact of GEM modest. Proteomic differential display analysis for GEM-sensitive human pancreatic adenocarcinoma cell line KLM1 and GEM-resistant KLM1-R cells by using two-dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry produced 33 protein spots. Of these, 23 were up-regulated and 10 were down-regulated in KLM1-R compared to KLM1 cells. The up-regulated proteins include acidic leucine-rich nuclear phosphoprotein 32 family member A, reticulocalbin-1, gamma-synuclein, microtubule-associated protein RP/EB family, sialic acid synthase, peptidyl-prolyl cis-trans isomerase A, far upstream element-binding protein 2 and catalase. The down-regulated proteins include far upstream element-binding protein 1, gamma-synuclein, galectin-1 and stathmin. Two spots of heat-shock protein 27 were up-regulated in KLM1-R cells. These results suggest an important complementary role for proteomics in the identification of proteins which may play a role in the poor response of pancreatic cancer to GEM.