The survival rate of patients with head and neck squamous cell carcinoma (HNSCC) stands at approximately 50% and this has not improved in decades. This study developed a novel sirtuin-3 (SIRT3) inhibitor (LC-0296) and examined its role in altering HNSCC tumorigenesis. The effect of the SIRT3 inhibitor, LC-0296, on cell survival, proliferation, and apoptosis, and reactive oxygen species levels in HNSCC cells were studied. LC-0296 reduces cell proliferation and promotes apoptosis of HNSCC cells but not of normal human oral keratinocytes. This inhibitory effect is mediated, in part, via modulation of reactive oxygen species levels. Additionally, LC-0296 works synergistically to increase the sensitivity of HNSCC cells to radiation and cisplatin treatment. Development of novel SIRT3 inhibitors, such as LC-0296, might enable the development of new targeted therapies to treat and improve the survival rate of patients with head and neck cancer.