BACKGROUND The precise molecular mechanisms underlying the gallbladder carcinoma (GBC) metastasis has not been fully elucidated. METHODS In the present study, metastasis-associated proteins were identified by comparative proteomic analysis. The functional study of the candidate protein vimentin was further investigated. First, a pair of higher and lower metastatic sublines (termed GBC-SD/M3 and GBC-SD, respectively), originated from the same parental cell line, was screened by spontaneous tumorigenicity and metastasis in vivo in animal study and further characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach comprised two-dimensional gel electrophoresis analysis and mass spectroscopy was used to identify and compare the protein expression patterns between higher metastatic GBC-SD/M3 and lower metastatic GBC-SD cell lines. Then twenty-six proteins were identified. RESULTS Among the 26 proteins identified, fourteen proteins were up-regulated and 12 proteins were down-regulated in GBC-SD/M3. Vimentin was identified and found to be overexpressed in GBC-SD/M3 as compared with GBC-SD. This result was further confirmed by quantitative PCR and Western blotting analysis. Furthermore, the cell migration and invasion potency of GBC-SD/M3 in vitro was remarkably suppressed after small interference RNA-mediated knockdown of vimentin. Moreover, immunoblot and immunohistochemical analysis on 12 human GBC specimens showed consistently increased vimentin expression in metastases compared with primary tumors. CONCLUSION Tumor vimentin level may reflect the pathological progression in some GBC and may be a useful marker for predicting tumor metastasis and a therapeutic target for the treatment of GBC patients with metastases.