Purpose: This study aimed to investigate the functional difference between hypoxia inducible factor (HIF)-1α and HIF-2α in oral squamous cell carcinomas (OSCC). Experimental Design: We evaluated the correlations between HIF-1α and HIF-2α expression and the clinical-pathologic characteristics of 97 patients with OSCC by immunohistochemical staining. OSCC cell lines transfected with lentivirus encoding short hairpin RNA against HIF-1α/2α were used to investigate the HIF-1α/2α- dependent target genes. Xenograft tumors in nude mice were established using cells affected by lentivirus, and tumor growth, angiogenesis, proliferation, and apoptosis were measured. Results: HIF-1α expression was significantly associated with T stage (P = 0.004), lymph node involvement (P = 0.006), histologic differentiation (P = 0.013), and microvessel density (P = 0.014), whereas that of HIF-2α was associated with T stage (P = 0.011) and microvessel density (P = 0.005). Patients with positive HIF-1α nuclear staining had a significantly worse overall survival (P < 0.001) and disease-free survival (P < 0.001) than those with negative HIF-1α staining. When OSCC cells were cultured at 5% O₂, only HIF-2α contributed to the expression of vascular endothelial growth factor. At 1% O₂, vascular endothelial growth factor was regulated by both HIF-1α and HIF-2α, but glucose transporter 1, carbonic anhydrase 9, and urokinase-type plasminogen activator receptor were regulated by HIF-1α rather than by HIF-2α. Knocking down HIF-1α or HIF-2α individually inhibited the xenograft tumor angiogenesis and growth, and knocking them down simultaneously revealed a better inhibitory effect than knocking down either unit alone. Conclusions: HIF-1α and HIF-2α correlated with different clinical-pathologic parameters, stabilized at different oxygen levels, and regulated different genes in OSCC. However, both HIF-1α and HIF-2α showed promoting roles in tumor angiogenesis and growth, and therapeutic outcome may benefit from combined targeting of HIF-1α and HIF-2α. ©2010 AACR.