Metastasis of squamous cell carcinoma of the head and neck (SCCHN) is a significant health-care problem worldwide. The 5-year survival rate is less than 50% for patients with lymph node metastases. Understanding the molecular basis of SCCHN metastasis would facilitate the development of new therapeutic approaches to the disease. To identify proteins that mediate SCCHN metastasis, we established a SCCHN xenograft mouse model and performed in vivo selection from a SCCHN cell line using the model. In the fourth round of in vivo selection, significant incidences of metastases in lymph nodes (7/10) and lungs (6/10) were achieved from a derived SCCHN cell line as compared with its parental cells, 1/5 in lymph nodes and 0/5 in lungs. Metastatic cell lines from lymph node metastases and parental cell lines from non-metastatic xenograft tumors were subjected to DNA microarray analysis using an Affymetrix gene chip HG-U133A, followed by data mining studies. The identified metastasis-related genes were further evaluated for their encoding protein products and the metastatic cells were examined by biological analyses. DNA microarray analysis highlighted molecular features of the metastatic SCCHN cells, including alteration of expression of cell-cell adhesion proteins, epithelial cell markers, apoptosis and cell cycle regulatory molecules. Further biological analyses of phenotypic alterations revealed that the metastatic cells gained epithelial-mesenchymal transition (EMT) features and were more resistant to anoikis, which are two of the important phenotypes for metastatic SCCHN.