© 2016 Wiley Periodicals, Inc. Background: Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (SCC) has improved clinical outcomes compared to HPV-negative disease. However, the biology underlying differences in prognosis remains unclear. Methods: We characterized the expression of DNA-protein kinase catalytic subunit (DNA-PkCS), a key DNA repair protein also associated with tumor progression, in 29 cases of oropharyngeal SCCs and correlated our findings with HPV status and disease recurrence. In addition, we assessed therapeutic response, migration, and invasion in head and neck cancer cell lines upon DNA-PkCS knockdown. Results: DNA-PkCS expression was significantly decreased in HPV-positive compared to HPV-negative oropharyngeal SCC samples. Within the HPV-positive subgroup, DNA-PkCS expression was inversely related to HPV E6 and E7 expression and trended toward significance as a predictor of recurrence. DNA-PkCS knockdown in cell lines resulted in increased sensitivity to cisplatin and radiotherapy and reduced cell migration and invasion. Conclusion: These results suggest DNA-PkCS should be further studied as a potential marker of tumor progression in HPV-positive oropharyngeal SCCs. © 2016 Wiley Periodicals, Inc. Head Neck 39: 206–214, 2017.