Lymphatic metastasis is the first step in the metastatic process of magligant tumors deriving from epithelia, and its uncertain mechanisms are always the major problem in the field of oncology. In order to obtain lymphatic metastasis-associated proteins, the protein expressed profiles of mouse hepatocarcinoma ascites syngeneic cell lines Hca-F with highly lymphatic metastatic potentiality and Hca-P with low lymphatic metastatic potentiality were compared using fluorescent two-dimensional difference gel electrophoresis(2D DIGE). DeCyde software was applied to analyze 2D DIGE images,and 163 differential protein spots between Hca-F and Hca-P were detected, including 86 up-regulated protein spots in Hca-F and 77 down-regulated protein spots in Hca-F. 23 protein sports representing differential ratio more than 2-fold were chosen to be analyzed by MS. 17 metastasis-associated proteins were identified, and the partial identified proteins were further validated by Western blotting analysis. Of the identified proteins, the expression of transketolase, vimentin, creatine kinase(brain), anxa7 protein, anxa5 protein,enoyl coenzyme A hydratase 1(peroxisomal), heterogeneous nuclear ribonucleoprotein A2/B1 isoform 1 were up-regulated in Hca-F. Whereas eukaryotic translation elongation factor 2, Ero1l, Aldh2 protein, malate dehydrogenase 2(NAD), lactamase beta 2, glutathione S-transferase omega1, ubiquitin carboxyl-terminal hydrolase isozyme L3, endoplasmic reticulum protein ERp29(precursor), lypla1, stathmin were down-regulated in Hca-F. On the basis of the Gene Ontology(GO)classification，the differential expression proteins were found to be involved in many of biological process, such as metabolism, protein secretion, protein binding, nucleic acid binding, calcium ion binding,apoptosis and regulation of growth etc. Validating the function of these proteins is helpful to elucidate the mechanisms of lymphatic metastasis.