Head and neck squamous cell carcinoma (HNSCC) is associated with a high rate of mortality and novel anti-HNSCC drugs must be developed. 8-Hydroxyquinoline derivatives are associated with anti-oxidant, anti-tumor and anti-HIV-1 properties. Tris(8-hydroxyquinoline)iron (Feq3), a novel compound, was synthesized. Feq3 induced the death of HNSCC cells and caused them to exhibit the morphological features of apoptosis. Feq3 also induced apoptosis of SCC9 cells by cell cycle arrest during the G2/M phase and the induced arrest of SCC25 cells in the G0/G1 and G2/M phases, which was associated with decreased cyclin B1/cdc2 and cyclin D/cdk4 expressions. Feq3 increases reactive oxygen species (ROS) and reduces glutathione (GSH) levels, and responds to increased p53 and p21 expressions. Feq3 induced apoptosis by mitochondria-mediated Bax and cytochrome c up-expression and downexpression Bcl-2. Feq3 also up-regulated tBid, which interacts with the mitochondrial pathway and tumor necrosis factor-α (TNF-α)/TNF-Rs, FasL/Fas, and TNF-related apoptosis inducing ligand receptors (TRAIL-Rs)/TRAIL-dependent caspases apoptotic signaling pathway in HNSCC cells. However, Feq3 activates Fas but not FasL in SCC25 cells. Feq3 arrests the growth of HNSCC cells and is involved in the mitochondria-and death receptor (DR)-mediated caspases apoptotic pathway. This study is the first to suggest that apoptosis mediates the anti-HNSCC of Feq3. Feq3 has potential as a cancer therapeutic agent against HNSCC.