Abstract: This study aims to elucidate the role of proline hydroxylase phd1 in hypoxia tolerance in zebrafish. To this end, wild-type (phd1^+/+) and phd1-deficient (phd1^-/-) zebrafish were subjected to hypoxic conditions. Then, erythrocyte numbers were analyzed using o-dianisidine staining, and hypoxia-responsive gene expression was assessed via real-time quantitative PCR. Additionally, the survival of zebrafish larvae and adults was measured. The study initially validated the conservation of the PHD1 protein in humans, mice, and zebrafish. Under hypoxic conditions, the number of erythrocytes in phd1^-/- zebrafish increased significantly compared to phd1^+/+ zebrafish, and phd1^-/- zebrafish larvae and adults exhibited enhanced tolerance to hypoxia. Furthermore, hypoxia-responsive genes were more strongly induced in phd1^-/- zebrafish than in phd1^+/+ zebrafish. These findings suggest that knocking out phd1 enhances hypoxia tolerance in zebrafish, advancing our understanding of the biological function of this gene in fish and offering a molecular target for breeding hypoxia-tolerant fish strains.