SAM pointed domain containing E26 transformation-specific transcription factor (SPDEF) plays dual roles in the initiation and development of human malignancies. However, the biological role of SPDEF in head and neck squamous cell carcinoma (HNSCC) remains unclear. In this study, the expression level of SPDEF and its correlation with the clinical parameters of patients with HNSCC were determined using TCGA-HNSC, GSE65858, and our own clinical cohorts. CCK8, colony formation, cell cycle analysis, and a xenograft tumor growth model were used to determine the molecular functions of SPDEF in HNSCC. ChIP-qPCR, dual luciferase reporter assay, and rescue experiments were conducted to explore the potential molecular mechanism of SPDEF in HNSCC. Compared with normal epithelial tissues, SPDEF was significantly downregulated in HNSCC tissues. Patients with HNSCC with low SPDEF mRNA levels exhibited poor clinical outcomes. Restoring SPDEF inhibited HNSCC cell viability and colony formation and induced G0/G1 cell cycle arrest, while silencing SPDEF promoted cell proliferation in vitro. The xenograft tumor growth model showed that tumors with SPDEF overexpression had slower growth rates, smaller volumes, and lower weights. SPDEF could directly bind to the promoter region of NR4A1 and promoted its transcription, inducing the suppression of AKT, MAPK, and NF-κB signaling pathways. Moreover, silencing NR4A1 blocked the suppressive effect of SPDEF in HNSCC cells. Here, we demonstrate that SPDEF acts as a tumor suppressor by transcriptionally activating NR4A1 in HNSCC. Our findings provide novel insights into the molecular mechanism of SPDEF in tumorigenesis and a novel potential therapeutic target for HNSCC.