Oxygen homeostasis is an important organizing principle for understanding development, physiology, disease, and evolution. Under various physiological and pathological states, organisms experience oxygen deficiency or hypoxia. FoxO4 has been recognized as an important transcriptional regulator involved in a variety of cellular functions, including proliferation, apoptosis, differentiation, and stress resistance, but its role in hypoxia adaptation mechanisms in animals is not so clear. To explore the role offoxO4in the hypoxia response, we detected the expression offoxO4and the regulatory relationship between Hif1α andfoxO4under hypoxic conditions. It was found that the expression offoxO4was up-regulated in ZF4 cells and zebrafish tissues after hypoxia treatment, and Hif1α could directly target the HRE of thefoxO4promoter to regulatefoxO4transcription, indicating thatfoxO4was involved in the hypoxia response by the Hif1α-mediated pathway. Furthermore, we obtainedfoxO4knockout zebrafish and found that the disruption offoxO4increased the tolerance to hypoxia. Further research found that the oxygen consumption and locomotor activity offoxO4−/−zebrafish were lower than those of WT zebrafish, as was true for NADH content, NADH/NAD+rate, and expression of mitochondrial respiratory chain complex-related genes. This suggests that disruption offoxO4reduced the oxygen demand threshold of the organism, which explained why thefoxO4−/−zebrafish were more tolerant to hypoxia than WT zebrafish. These results will provide a theoretical basis for further study of the role offoxO4in the hypoxia response.