Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide. The majority of HNSCCs overexpress Epidermal Growth Factor Receptor (EGFR), an essential receptor tyrosine kinase (RTK) that promotes HNSCC growth and metastasis. Therefore, EGFR has been used as an important therapeutic target to treat HNSCC. Inhibition of EGFR stimulates autophagy in cancer cells. However, the role of autophagy in EGFR inhibitor-induced cancer suppression is still in a debate. Here, we reveal that the first-and the second-generation EGFR tyrosine kinase inhibitors (TKIs) differentially affect HNSCC autophagy. The second-generation EGFR TKIs have much stronger effects on autophagy than the first-generation TKIs. The second-generation EGFR TKIs not only promote autophagy initiation signaling but also block autophagic flux by disturbing the lysosomes function, indicating a novel mechanism by which EGFR TKIs modulate cancer cell autophagy. Blocking the initiation of autophagy does not affect the second-generation EGFR TKIs-induced HNSCC growth suppression. This suggests that the anti-growth effect of the second-generation EGFR TKIs on HNSCC is not dependent on autophagy. (C) 2017 Elsevier Inc. All rights reserved.