Multifaceted and Intricate Oncogenic Mechanisms of NDRG1 in Head and Neck Cancer Depend on Its C-terminal 3R-Motif

N-Myc downstream-regulated 1 (NDRG1) has inconsistent oncogenic functions in various cancers. We surveyed and characterized the role of NDRG1 in head and neck cancer (HNC). Cellular methods included spheroid cell formation, clonogenic survival, cell viability, and Matrigel invasion assays. Molecular techniques included transcriptomic profiling, RT-qPCR, immunoblotting, in vitro phosphorylation, immunofluorescent staining, and confocal microscopy. Prognostic significance was assessed by Kaplan–Meier analysis. NDRG1 participated in diverse oncogenic functions in HNC cells, mainly stress response and cell motility. Notably, NDRG1 contributed to spheroid cell growth, radio-chemoresistance, and upregulation of stemness-related markers (CD44 and Twist1). NDRG1 facilitated cell migration and invasion, and was associated with modulation of the extracellular matrix molecules (fibronectin, vimentin). Characterizing the 3R-motif in NDRG1 revealed its mechanism in the differential regulation of the phenotypes. The 3R-motif displayed minimal effect on cancer stemness but was crucial for cell motility. Phosphorylating the motif by GSK3b at serine residues led to its nuclear translocation to promote motility. Clinical analyses supported the oncogenic function of NDRG1, which was overexpressed in HNC and associated with poor prognosis. The data elucidate the multifaceted and intricate mechanisms of NDRG1 in HNC. NDRG1 may be a prognostic indicator or therapeutic target for refractory HNC.