Gain-of-function mutations of KIT are common genetic events in gastrointestinal stromal tumors (GISTs). To investigate the molecular characteristics of KIT mutations in GISTs, 20 GISTs (14 GISTs with KIT mutation and 6 GISTs without KIT mutation) were analyzed by two-dimensional electrophoresis and matrix-associated laser desorption ionization mass spectrophotometry-time of flight. Comparative analysis of the respective spot patterns on two-dimensional electrophoresis showed that HMGB1, an intranuclear protein that interacts with several transcription factors and plays a role in tumor metastasis after its secretion, was overexpressed in GISTs with KIT mutation. All of the 14 GISTs with KIT mutation, and only 2 of 6 GISTs without KIT mutation, revealed HMGB1 expression. Of the GISTs with KIT mutation, 12 (86%) showed strong expression of HMGB1, more than three times higher in intensity than the maximum observed in the 6 GISTs without KIT mutation by two-dimensional electrophoresis analysis. The overexpression of HMGB1 was further supported by Western blot analysis, and directly related to matrix metalloproteinase 2 overexpression. Our results indicate that the overexpression of HMGB1 is common in GISTs and is related to the KIT mutation, and that this may play a role in the tumorigenesis of GISTs because overexpressed HMGB1 could accelerate genes related to tumor growth and invasion.