The transcription factor hypoxia inducible factor-1alpha (HIF-1alpha) mediates adaptive responses to hypoxia by nuclear translocation and regulation of gene expression. Mitochondrial changes are critical for the adaptive response to hypoxia. However, the transcriptional and non-transcriptional mechanisms by which HIF-1alpha regulates mitochondria under hypoxia are poorly understood. Here, we examined the subcellular localization of HIF-1alpha in human cells and identified a small fraction of HIF-1alpha that translocated to the mitochondria after exposure to hypoxia or hypoxia-mimicking pharmacological agents. To probe the function of this HIF-1alpha population, we ectopically expressed a mitochondrial-targeted form of HIF-1alpha (mito-HIF-1alpha). Expression of mito-HIF-1alpha was sufficient to attenuate apoptosis induced by exposure to hypoxia or H2O2-induced oxidative stress. Moreover, mito-HIF-1alpha expression reduced the production of reactive oxygen species, the collapse of mitochondrial membrane potential, and the expression of mitochondrial DNA-encoded mRNA in response to hypoxia. However, these functions of mito-HIF-1alpha were independent of its conventional transcriptional activity. Finally, the livers of mice with CCl4-induced fibrosis showed a progressive increase in HIF-1alpha association with the mitochondria, indicating the clinical relevance of this finding. These data suggested that mitochondrial HIF-1alpha protects against apoptosis independently of its well-known role as a transcription factor.