Laryngeal Squamous Cell Carcinoma (LSCC) is one of the most common malignancy in Head and neck cancer for which the mechanism underlying its metastasis is poorly understood. Myosin X, a molecular motor in cells has been demonstrated to play an important role in cell migration. However, whether Myosin X is involved in the metastasis of LSCC remains unclear. To investigate the expression of Myosin X and its implication in the metastasis of LSCC, we recruited 30 patients with LSCC and 6 patients with vocal cord polyp range from October 2016 to October 2018. Tissue samples were obtained during surgery and the expression of Myosin X, Cortactin, MMP2, MMP9, E-cadherin, and β-catenin in tissue samples were evaluated by RT-PCR, Western blot, immunohistochemistry or ELISA. Patients with LSCC were further followed-up 2 year after surgery for metastasis analysis. We found that the level of Myosin X, Cortactin, MMP2, and MMP9 was much higher in poorly differentiated LSCC compared to that in moderately and highly LSCC, as well as the control tissues. In contrast, the expression of epithelial-mesenchymal transition related marker, E-cadherin, and β-catenin, were much lower in poorly differentiated LSCC tissues compared to that in moderately and highly differentiated LSCC tissues, as well as the control tissues. Moreover, the expression of Myosin X was positively correlated with Cortactin, MMP2, and MMP9 levels. Increased expression of Myosin X in LSCC tissues was related to higher risk of metastasis. In conclusion, our findings showed that. Myosin X augments the expression of Cortactin, MMP2 and MMP9, which could upregulate the cell migration and the matrix degradation, and consequently reduce the expression of E-cadherin and β-catenin, thereby activating epithelial-mesenchymal transformation and promoting the metastasis of LSCC. Targeting Myosin X may have potential therapeutic effect in the metastasis of LSCC.