Head and neck squamous cell carcinoma (HNSCC) is a world-wide malignancy. This study aimed to identify differential gene expression associated with the progression of disease from primary to metastatic HNSCC. Microdissection retrieved pure epithelial cells from paired primary tumours and cervical lymph node metastasis. cDNA microarray analysis and algorithm grouping identified differential mRNA expression of 301 genes. Quantitative reverse transcription-polymerase chain reaction analysis clarified the up-regulation of CCL19, CR2, EGR2, FUCA1, RGS1, and SELL, as well as the down-regulation of IGFBP6 and KLK8 in nodal metastasis compared to primary tumours. Immunohistochemistry confirmed the up-regulation of SELL and down-regulation of IGFBP6 in nodal metastasis relative to primary tumours. Interestingly, primary tumours exhibiting higher FUCA1 and SELL expression were associated with significantly worse patient survival. In OECM-1 HNSCC cells, inhibition of proliferation, migration, and anchorage-independent growth was noted following knockdown of SELL expression. In SAS HNSCC cells, expression of exogenous SELL resulted in increased invasion, anchorage-independent growth, and xenographic tumourigenesis in nude mice. Knockdown of FUCA1 and treatment with IGFBP6 inhibited the migration of OECM-1 cells. Knockdown of RGS1 inhibited the anchorage-independent growth of SAS cells. Our results provide a useful gene signature profile describing the factors underlying the metastasis of HNSCC to cervical lymph nodes, which may be beneficial for the treatment of HNSCC metastasis.