Objectives: The aim of the present study was to explore the functional role of heparanase (HPSE) and investigate the effect of HPSE on epithelial-mesenchymal transition (EMT) and Tumor-infiltrating activated natural killer cells in oral squamous cell carcinoma (OSCC).Materials and methods: human oral squamous carcinoma (SCC-25) cells were transfected with HPSE-specific small interfering RNA. Cell Counting Kit-8 assay was performed to examine cell proliferation, while flow cytometry was performed to analyze the cell cycle. Scratch assay was conducted to analyze cell migration, followed by Transwell assay to determine cell invasion. Real-Time Polymerase Chain Reaction and Western-blot assays were performed to measure epithelial-mesenchymal transition protein expression. RNA Sequencing analysis and tumor-infiltrating immune cells estimation were performed to elucidate the effect of HPSE on OSCC.Results: Knockdown of HPSE expression decreased the proliferation rate of SCC-25 cells resulting in a significant elevation in cell percentage at the Gap phase 0/Gap phase 1 phase by suppressed cell migration and invasion. The E-cadherin messenger RNA and protein expression increased while Snail and Vimentin expression decreased. RNA Sequencing analysis performed between small interfering RNA and negative control groups identified 42 differentially expressed genes, such as syndecan binding protein, RAB11A, member RAS oncogene family, and DDB1 and CUL4 associated factor 15. Conclusions: These results indicated that knockdown of HPSE suppressed SCC-25 cell proliferation, invasion, migration, and epithelial-mesenchymal transition, possibly via syndecan binding protein and RAB11A, member RAS oncogene family. Moreover, HPSE regulates the infiltrated levels of natural killer cells activated, possibly via DDB1 and CUL4 associated factor 15.