With the rapid growth of human population and economy, the problem of eutrophication has become worse in the recent years. Hypoxia caused by eutrophication has been one of the main factors that restrict the continuing development of aquiculture. In this study, a subtractive cDNA library was successfully constructed with total RNA made from goldfish blastulae embryonic (CAB) cells treated with 1% O2 for 24 h and control cells using suppression subtractive hybridization (SSH). From this subtractive cDNA library, 1117 ESTs were identified through randomly screening 1300 colonies by PCR. Sequencing analysis revealed 260 genes in which 38 genes had been found to be hypoxia-induced genes in mammals and 208 genes were firstly reported to be hypoxia-responsive genes in fish. We noted that heme oxyenase-1 was induced greatly by hypoxia when selected 13 genes of all for hypoxia-induced analysis using semi-quantitive RT-PCR. So, we aimed to study this gene under hypoxic stress. In this report, a cDNA encoding Carassius auratus HO-1 (CaHO-1) was isolated from an SSH cell line, C. auratus blastulae embryonic (CAB) cells, exposed to 1% O2 for 24h. The CaHO-1 cDNA is 1247bp in length that encodes 272 amino acid residues. CaHO-1 is homologous to mammalian HO-1 in the primary structure, characterized by heme oxygenase domain, a heme oxygenase signature motif and a putative trans-membrane domain. Phylogenetic analysis shows that CaHO-1 had the highest homology with zebrafish HO-1 in amino acid level. RT-PCR and real-time PCR analysis indicated that CaHO-1 was predominantly transcribed in posterior kidney, head kidney, gill and intestine, and significantly predominant induction expression was observed in posterior kidney under hypoxic stress. Moreover, the significantly hypoxia-induced expression was confirmed in goldfish larvae and in the in vitro cultured CAB cells. Green fluorescence of HO-1-GFP fusion protein revealed its cytoplasm and plasma membrane localization, which was consistent with the putative trans-membrane structure. Subsequently, we established stably transfected CAB/pcDNA3.1-HO-1 cell line and control CAB/pcDNA3.1 cell line, and found that the death cells were obviously reduced in the pcDNA3.1-HO-1 transfected cells under 4 days of hypoxia (1% O2) treatments in comparison with numerous detached death cells in the control pcDNA3.1 transfected cells. Furthermore, a significant cell viability difference between two kinds of transfected cells during hypoxia treatment and reoxygenation was revealed by using Cell Counting Kit-8 for cell viability assay. Therefore, the data suggested that fish HO-1 might play a significant protection role for the cells in response to hypoxic stress.