Lymph node (LN) metastasis is one of the most important adverse prognostic factors for pancreatic cancer. The aim of this study was to identify novel lymphatic metastasis-associated markers for pancreatic cancer. DNA microarray analysis was used to determine and compare the expression profiles of 17 pancreatic cancer tissues with LN metastasis and 17 pancreatic cancer tissues without LN metastasis. The microarray results were validated by real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Only 58 genes were differentially expressed between the two groups with a difference in signal intensity ratio greater than a 1.5-fold change. Of these genes, 30 were significantly down-regulated in the LN metastasis group. Among five selected down-regulated genes for validation using real-time PCR, the expression of DST, FosB, RGS16, and CXCL12 was significantly lower in the LN metastasis group. Immunohistochemical analysis confirmed RGS16 and FosB underexpression in pancreatic cancer tissues with LN metastasis. RGS16 and FosB underexpression was associated with poor patient survival. Our findings show that RGS16 and FosB are underexpressed in pancreatic cancer with lymph node metastasis and associated with reduced survival, suggesting that RGS16 and FosB might be prognostic markers for pancreatic cancer.