Background: Most human tumors develops regions of chronically or transient hypoxic cells during growth. The effect of hypoxia on tumor tissue has a strong impact on tumor cell migration and metastatic behavior. uPA a major member plasminogen activator system that is involved at different stages of metastasis. Aim: To compare uPA expression in three oral tongue squamous cell carcinoma (OTSCC) and normal human oral keratinocytes (NHOK) cell lines and study the effect of hypoxia on that expression. Methods: Three Oral Tongue Squamous Cell Carcinoma (OTSCC) and three Normal Human Oral Keratinocytes (NHOK) cell lines were analyzed under normoxia and hypoxia for uPA mRNA concentration with northern blot analysis and uPA protein expression with western blot analysis, the expressed protein was further examined for active component with fibrin overlay zymography, and immunocytochemistry were utilized to confirm uPA expression. Results: uPA was expressed under normoxia condition in all three OTSCC and NHOK cell lines with much higher basal level in tongue cancer. Hypoxia highly upregulated uPA mRNA expression in OTSCC compared to NHOK (P= 0.00012) and uPA protein as well (P= 0.029), and active uPA was also upregulated (P= 0.008). Conclusion: uPA genetic expression is an important regulator for metastatic behavior and invasiveness of oral tongue squamous cell carcinoma (OTSCC), and hypoxia plays an essential role for its upregulation.