The levels of hypoxia-inducible factor 1α (HIF-1α) are tightly controlled. Here, we investigated the posttranscriptional regulation of HIF-1α expression in human cervical carcinoma HeLa cells responding to the hypoxia mimetic CoCl₂. Undetectable in untreated cells, HIF-1α levels increased dramatically in CoCl₂-treated cells, while HIF-1α mRNA levels were unchanged. HIF-1α translation was potently elevated by CoCl₂ treatment, as determined by de novo translation analysis and by monitoring the polysomal association of HIF-1α mRNA. An internal ribosome entry site in the HIF-1α 5′ untranslated region (UTR) was found to enhance translation constitutively, but it did not further induce translation in response to CoCl₂ treatment. Instead, we postulated that RNA-binding proteins HuR and PTB, previously shown to bind HIF-1α mRNA, participated in its translational upregulation after CoCl₂ treatment. Indeed, both RNA-binding proteins were found to bind HIF-1α mRNA in a CoCl₂-inducible manner as assessed by immunoprecipitation of endogenous ribonucleoprotein complexes. Using a chimeric reporter, polypyrimidine tract-binding protein (PTB) was found to bind the HIF-1α 3′UTR, while HuR associated principally with the 5′UTR. Lowering PTB expression or HuR expression using RNA interference reduced HIF-1α translation and expression levels but not HIF-1α mRNA abundance. Conversely, HIF-1α expression and translation in response to CoCl₂ were markedly elevated after HuR overexpression. We propose that HuR and PTB jointly upregulate HIF-1α translation in response to CoCl₂.