Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing protein with antioxidant properties. GPx-3 deficiency has been associated with cardiovascular disease and stroke. The regulation of GPx-3 expression remains largely uncharacterized, however, and we studied its transcriptional and translational determinants in a cultured cell system. In transient transfections of a renal cell line (Caki-2), the published sequence cloned upstream of a luciferase reporter gene produced minimal activity (relative luminescence (RL) = 0.6 ± 0.4). Rapid amplification of cDNA ends was used to identify a novel transcription start site that is located 233 bp downstream (3′) of the published site and that produced a >25-fold increase in transcriptional activity (RL = 16.8 ± 1.9; p < 0.0001). Analysis of the novel GPx-3 promoter identified Sp-1- and hypoxia-inducible factor-1-binding sites, as well as the redox-sensitive metal response element and antioxidant response element. Hypoxia was identified as a strong transcriptional regulator of GPx-3 expression, in part through the presence of the hypoxia-inducible factor-1-binding site, leading to an almost 3-fold increase in expression levels after 24 h compared with normoxic conditions (normalized RL = 3.5 ± 0.3 versus 1.2 ± 0.1; p < 0.001). We also investigated the role of the translational cofactors tRNASec, SECIS-binding protein-2, and SelD (selenophosphate synthetase D) in GPx-3 protein expression. tRNASec and SelD significantly enhanced GPx-3 expression, whereas SECIS-binding protein-2 showed a trend toward increased expression. These results demonstrate the presence of a novel functional transcription start site for the human GPx-3 gene with a promoter regulated by hypoxia, and identify unique translational determinants of GPx-3 expression.