AbstractHead and neck squamous cell carcinoma (HNSCC) has a variety of causes. Recently, the human papilloma virus (HPV) has been implicated in the rising incidence of oropharyngeal cancer and has led to variety of studies exploring the differences between HPV-positive and HPV-negative HNSCC. The calcium-activated chloride channel TMEM16A is overexpressed in a variety of cancers, including HNSCC, but whether or not it plays different roles in HPV-positive and HPV-negative HNSCC is unknown. Here, we demonstrate that TMEM16A is preferentially overexpressed in HPV-negative HNSCC and that this overexpression of TMEM16A is associated with decreased patient survival. We also show that TMEM16A expression is decreased in HPV-positive HNSCC at the DNA, RNA and protein levels in patient samples as well as cell lines. We demonstrate that the lower levels of TMEM16A expression in HPV-positive tumors can be attributed to both a combination of copy number alteration and promoter methylation at the DNA level. Additionally, our cellular data show that HPV-negative cell lines are more dependent on TMEM16A for survival than HPV-positive cell lines. Therefore, we suspect that the down-regulation of TMEM16A in HPV-positive HNSCC makes TMEM16A a poor therapeutic target in HPV-positive HNSCC, but a potentially useful target in HPV-negative HNSCC.