〔Abstract〕 Objective To investigate the biological role of aqueous-soluble components of sporoderm-removed Ganoderma lucidum spore powder(A-GSP) in head and neck squamous cell carcinoma(HNSCC). Methods The effects of A-GSP on the proliferation and metastasis of head and neck squamous cell carcinoma cells SCC15 and SCC25 were investigated using MTS, plate cloning, scratch healing experiments, and chamber penetration experiments. The effect of A-GSP on the apoptosis ability and cell cycle distribution of head and neck squamous cell carcinoma cells was detected using a flow cytometry assay kit. Western blot was used to detect the expression changes of epithelial mesenchymal transformation(EMT) molecules and cell cycle proteins after A-GSP treatment. Results After the action of A-GSP, the proliferation ability of head and neck squamous cell carcinoma cells decreased(F=62.72, P<0.000 1), the metastasis ability decreased(F=95.7, P<0.001), the apoptosis ability increased(F=87.65, P<0.001), and the G2/M phase stagnated(F=15.31, P<0.01). In addition, A-GSP treatment resulted in upregulation of epithelial cell markers E-cadherin, downregulation of mesenchymal cell markers Vimentin, N-cadherin, Snail, Slug, downregulation of cell cycle kinase CDK1, and upregulation of P21 and P27. However, it did not cause changes in the levels of apoptosis pathway proteins Caspase and Bax. Conclusion A-GSP inhibits the proliferation and metastasis of head and neck squamous cell carcinoma cells by inducing G2/M phase arrest and inhibiting EMT.