The ubiquitously expressed hypoxia-inducible factor-1α (HIF-1α) acts as a key transcription factor in regulating metabolism, development, cellular survival, proliferation and pathology under hypoxia condition. Compared to mammals, fish are more vulnerable to hypoxia stress and contamination; however, the regulation of HIF-1α in fish remains obscure. In this study, zebrafish HIF-1α promoter was cloned and found to possess a CpG island located at -97 to +403, but the canonical TATA-box was absent. Aligning 240-bp HIF-1α proximal promoter region of zebrafish with other vertebrates showed more than 82 % identity with cyprinid fishes. Further luciferase analysis suggested that the minimal core promoter might locate at -134 to +97, and several putative transcription factor binding sites were found in this region by bioinformatic analysis. Moreover, it was shown that the zebrafish HIF-1α mRNA was significantly activated by 10 μg/mL lipopolysaccharide (LPS) under hypoxia condition and peaked at 8 h after treatment, suggesting LPS- and hypoxia-regulated zebrafish HIF-1α transcriptional activity in a synergistic pattern. This synergistic effect was closely related to the living environment of fish, indicating that this mechanism would be more conducive to fish survival.