Autoimmune pancreatitis (AIP) is defined by characteristic lymphoplasmacytic infiltrate, ductal strictures and a pancreatic enlargement or mass that can mimic pancreatic cancer (PaCa). The distinction between this benign disease and pancreatic cancer can be challenging. However, an accurate diagnosis may pre-empt the misdiagnosis of cancer, allowing the appropriate medical treatment of AIP and, consequently, decreasing the number of unnecessary pancreatic resections. Mass spectrometry (MS) and two-dimensional differential gel electrophoresis (2D-DIGE) have been applied to analyse serum protein alterations associated with AIP and PaCa, and to identify protein signatures indicative of the diseases. Patients' sera were immunodepleted from the 20 most prominent serum proteins prior to further 2D-DIGE and image analysis. The identity of the most-discriminatory proteins detected, was performed by MS and ELISAs were applied to confirm their expression. Serum profiling data analysis with 2D-DIGE revealed 39 protein peaks able to discriminate between AIP and PaCa. Proteins were purified and further analysed by MALDI-TOF-MS. Peptide mass fingerprinting led to identification of eleven proteins. Among them apolipoprotein A-I, apolipoprotein A-II, transthyretin, and tetranectin were identified and found as 3.0-, 3.5-, 2-, and 1.6-fold decreased in PaCa sera, respectively, whereas haptoglobin and apolipoprotein E were found to be 3.8- and 1.6-fold elevated in PaCa sera. With the exception of haptoglobin the ELISA results of the identified proteins confirmed the 2D-DIGE image analysis characteristics. Integration of the identified serum proteins as AIP markers may have considerable potential to provide additional information for the diagnosis of AIP to choose the appropriate treatment.