Cancer cell metastasis is the major cause of lung adenocarcinoma (AdC) with high mortality and poor prognosis. To screen metastasis-associated biomarkers of lung AdC, laser capture microdissection (LCM) was used to purify the cancer cells from primary lung AdC with (LNM AdC) and without metastasis (non-LNM AdC) according to clinical diagnosis of lymph node metastasis and distant metastasis. Then two-dimensional differential in-gel electrophoresis (2D-DIGE) was performed to isolate the total proteins of the pooled microdissected cancer cells from non-LNM AdC and LNM AdC. The differential proteins between non-LNM AdC and LNM AdC were analyzed by Decyder software and further identified by mass spectrometry (MS). The partial differential proteins annexin A1, annexin A2, annexin A3, S100A9 and B23 were validated by Western blot. 2D-DIGE patterns of microdissected non-LNM AdC and LNM AdC were established, and 20 differential proteins in the above two tissues were identified, of which 13 proteins were up-regulated and 7 proteins were down-regulated in LNM AdC compared to non-LNM AdC. Western blot results indicated that annexin A1, annexin A2, annexin A3 and S100A9 were significantly up-regulated in LNM AdC compared to non-LNM AdC; B23 was significantly down-regulated in LNM AdC compared to non-LNM AdC. Immunohistochemical analysis indicated S100A9 was up-regulated in LNM AdC compared with non-LNM AdC. It was the first time that metastasis-associated proteins were identified in primary adenocarcinoma by LCM coupled with 2D-DIGE and MS techniques. The findings will facilitate understanding of lung AdC metastasis and provide some direct proof for mining markers for predicting metastasis and patients’ outcome so as to improve the diagnose and treatment of lung AdC.