Background. Hypoxia-inducible factor-1α (HIF-1α), a subunit of the HIF-1 protein, plays a key role in the regulation of genes involved in hypoxia physiological response. Tongue sole, Cynoglossus semilaevis Günther, 1873, a marine teleost, had been proved to be a hypoxia-tolerant species. In this study, the HIF-1α in tongue sole was cloned and its expression under acute hypoxia was examined to provide further basis for understanding the molecular response of tongue sole under hypoxia. Materials and Methods. The full length of HIF-1α cDNA sequence was cloned from the liver of tongue sole by RT-PCR and RACE-PCR method. Then, the expression pattern of tongue sole HIF-1α under acute hypoxic conditions were detected using quantitative real-time PCR method. Results. The open reading frame of tongue sole HIF-1α is 2208 bp, encoding 735 amino acids. The amino acid sequence of tongue sole HIF-1α shared high identities (52.7%–81.8%) with HIF-1α from other vertebrates, and possessed six typical domains of the HIF-1 family (bHLH, PAS-A, PAS-B, PAC, N-TAD, and C-TAD). In adult fish, HIF-1α mRNAs were highly expressed in the liver, moderately in the heart, spleen, kidney, stomach, blood and gills, and low in intestine. Under acute hypoxia stress, expression of HIF-1α mRNAs were significantly up-regulated in many tissues, including the liver, spleen, stomach, blood, heart and gills. Conclusion. Tongue sole HIF-1α possessed the similar sequence length, shared higher identities and clustered well with other known HIF-1α, thus revealing a high degree conservation of HIF-1α during evolution. Tongue sole HIF-1α began to be up-regulated from 5 min to 120 min after hypoxia, indicating that it might play a significant role at the early stage of hypoxia.