Hypoxia-inducible factor-α (HIF-1α) is the key transcription factor involved in the adaptive response to hypoxia. It has been established that HIF-1α is overexpressed in various human cancers, including oral squamous cell carcinoma (OSCC), and orchestrates a wide spectrum of many key cancer molecular pathways involved in diverse tumor progression. In this study, RNA interference (RNAi) was introduced to downregulate the expression of HIF-1α in OSCC in vitro and in vivo. The mRNA and protein expression levels of HIF-1α and vascular epidermal growth factor were detected by quantitative real-time reverse transcription-PCR, western blot, and immunohistochemistry, and cell proliferation activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Tumor growth, microvascular density, apoptosis, and the expression of proliferation-related gene Ki-67 in tumor xenografts were evaluated. Our data demonstrated that small interfering RNA targeting HIF-1α significantly inhibited the mRNA and protein expression levels of HIF-1α and vascular epidermal growth factor. In addition, marked suppression of tumor cell prolifeation and xenograft growth, significant microvascular density suppression, reduced expression of Ki-67, and increased cell apoptosis were observed. These findings suggest that RNAi targeting HIF-1α could effectively inhibit the progression of OSCC. Thus, it may be used as a potent and specific therapy for oral cancer, especially in inhibiting and preventing cancer cell angiogenesis and survival.