The aim of the study was to observe different protein profiles in pancreatic cancer with and without lymph node metastasis (LNM), and search for novel LNM-associated proteins, which would help to understand the metastatic mechanisms and provide targets for therapeutic interventions. Cancer nests were manually miscrodissected from 8 LNM and 7 non-LNM pancreatic cancer tissues, and the protein extracts were then separated by difference gel electrophoresis (DIGE) and identified by MALDI-TOF-TOF. Four differently regulated proteins, ezrin, radixin, moesin, and c14orf166, were selected for further validation by Western blot and immunohistochemistry. In DIGE analysis, we identified 18 up-regulated proteins and 15 down-regulated proteins in LNM pancreatic cancer nests compared with non-LNM ones. Western blot and immunohistochemical analyses confirmed that radixin, moesin and c14orf166, but not ezrin, had significantly higher expression levels in LNM pancreatic cancers than in non-LNM controls. In conclusion, the specific protein profiles found in this study might provide new insights into the mechanism of lymph node metastasis. For the first time, c14orf166 was identified asa novel metastasis-associated protein, and the roles of radixin, moesin and c14orf166 in cancer metastasis deserve further investigations.