Laryngeal squamous cell carcinoma (LSCC) exhibits aggressive growth, frequent recurrence, and a notable resistance to existing treatments. Building upon prior discoveries that identified junctional adhesion molecule 3 (JAM3) as a critical tumor suppressor in LSCC, this study delves into the transcriptional regulation by upstream stimulatory factor 1 (USF1) and its implications for LSCC pathogenesis. Employing dual-luciferase assays and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR), we confirmed USF1's direct binding to the E-box within the JAM3 promoter, thereby enhancing JAM3 expression in AMC-HN-8 and FD-LSC-1 cells. Complementary in vitro assays and in vivo experiments corroborated that USF1 overexpression markedly reduces tumor aggressiveness, linked to heightened JAM3 activity. Further analysis, including Western blot and immunohistochemistry of xenograft tumor tissues, revealed that increased JAM3, stimulated by USF1, activates the Hippo signaling pathway, underscoring its role in tumor suppression. These findings position USF1 and JAM3 as pivotal elements in the molecular framework of LSCC, suggesting their potential as targets for therapeutic intervention.