Resistance to targeted therapies such as the EGFR tyrosine kinase inhibitors (TKIs), which is often associated with epithelial to mesenchymal transition (EMT), is a major issue in cancer treatment. In head and neck cancer (HNC), acquired resistance to anti-EGFR therapeutics is common and limits survival. To investigate these mechanisms, we established a model of acquired resistance to the EGFR TKI erlotinib in HNC HN5 cells. The erlotinib-resistant cells (ER-HN5) underwent EMT with increased vimentin and N-cadherin and reduced E-cadherin expression, respectively. The ER-HN5 cells had reduced EGFR signalling and activity, but increased Akt activity - a critical effector of the EGFR signalling pathway. ER-HN5 cells were more migratory and demonstrated increased expression and activity of Axl. Axl is a tyrosine kinase receptor that promotes invasion, migration, proliferation and resistance to targeted therapeutics and when overexpressed, is associated with tumour progression and a poor prognosis. Array analysis of the ER-HN5 cells revealed upregulation of Axl, as well as changes in expression of IL-6, IL-8 and a number of key signalling proteins compared to parental cells. ER-HN5 cells were sensitive to treatment with the Axl inhibitor R428, which is currently in preclinical testing for other tumours. Treatment with R428 also inhibited migration of ER–HN5 cells and the combination of erlotinib and R428 significantly reduced Akt activity as well as sensitising ER-HNC cells to a sub-effective dose of erlotinib (p<0.0001). Taken together, these data demonstrate a central role for Axl in acquired EGFR TKI resistance in HNC, and suggest that Axl inhibitors may help restore sensitivity of these resistant tumours to TKIs.