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Background Primary gallbladder carcinoma (PGC) is very lethal because of its early metastasis, strong invasion and poor prognosis.Data are limited regarding consistent molecular changes associated with gallbladder carcinogenesis and disease progression.Objective To find biomarkers which are involved in the development and progression of PGC.Methods Fresh PGC, cholecystitis and normal gallbladder tissues from 10 patients respectively were subjected to a comparative proteomic analysis.The proteomic patterns of PGC samples were compared to cholecystitis and normal gallbladder tissues using two-dimensional gel electrophoresis.And the differentially expressed proteins were selected and identified by MALDI-TOF-MS and database search.For validation experiments, four paired gallbladder cancer tissues and normal tumor-adjacent tissues was collected for immunoblotting analysis of ANXA4.Immunohistochemical staining of ANXA4 was performed on a tissue microarray containing 59 PGC tissues, 7 normal gallbladder tissues and 6 cholecystitis specimens to further investigate the clinical significance of the