Hypoxia-inducible factor-1alpha (HIF-1alpha) is a central regulator that controls the hypoxic response of mammalian cells through the induction of various target genes, and its expression contributes to the development and malignant progression of many tumors. We previously reported that some chemotherapeutic drugs and gamma-rays induce HIF-1alpha expression through increased production of reactive oxygen species (ROS) in oral squamous cell carcinoma (OSCC) cells. However, the mechanism by which intracellular ROS activate HIF-1alpha expression is poorly understood. In this study, we investigated the influence of ROS on HIF-1alpha signaling in OSCC cells by the transfection of manganese-superoxide dismutase (Mn-SOD)-specific small interfering RNA (siRNA). The levels of HIF-1alpha protein and mRNA were increased by siRNA under both normoxic and hypoxic conditions in parallel with the increase in intracellular ROS levels. The accumulation of HIF-1alpha protein was enhanced through inhibition of the recruitment of von Hippel-Lindau protein and HIF-1alpha ubiquitination without a change in prolyl hydroxylase mRNA and protein levels. Furthermore, the transactivation of HIF-1alpha was enhanced via cap-dependent and internal ribosome entry site-mediated mechanisms. These results suggest that intracellular ROS produced by the knockdown of Mn-SOD enhance HIF-1alpha expression in OSCC cells through transcriptional, translational, and posttranslational regulation.