Head and neck squamous cell carcinoma (HNSCC) remains one of the most common malignancies worldwide. Although the treatment outcomes of HNSCC have improved in recent years, the prognosis of patients with advanced-stage disease remains poor. Current treatment strategies for HNSCC include surgery as a primary therapy, while radio-, chemo-, and biotherapeutics can be applied as second-line therapy. Although tumor necrosis factor-α (TNF-α) is a potent tumor suppressor cytokine, the stimulation of opposing signals impairs its clinical utility as an anticancer agent. The aim of this study was to elucidate the mechanisms regulating TNF-α‑induced opposing signals and their biological consequences in HNSCC cell lines. We determined the molecular mechanisms of TNF-α-induced opposing signals in HNSCC cells. Our in vitro analysis indicated that one of these signals triggers apoptosis, while the other induces both apoptosis and cell survival. The TNF-α-induced survival of HNSCC cells is mediated by the TNF receptor-associated factor 2 (TRAF2)/nuclear factor (NF)-κB-dependent pathway, while TNF-α-induced apoptosis is mediated by mitochondrial and non-mitochondrial-dependent mechanisms through FADD-caspase-8-caspase-3 and ASK-JNK-p53-Noxa pathways. The localization of Noxa protein to both the mitochondria and endoplasmic reticulum (ER) was found to cause mitochondrial dysregulation and ER stress, respectively. Using inhibitory experiments, we demonstrated that the FADD‑caspase-8‑caspase-3 pathway, together with mitochondrial dysregulation and ER stress-dependent pathways, are essential for the modulation of apoptosis, and the NF-κB pathway is essential for the modulation of anti-apoptotic effects/cell survival during the exposure of HNSCC cells to TNF-α. Our data provide insight into the mechanisms of TNF-α-induced opposing signals in HNSCC cells and may further help in the development of novel therapeutic approaches with which to minimize the systemic toxicity of TNF-α.