Abstract: To investigate the function of prolyl hydroxylase domain-containing protein 3 (PHD3) in the hypoxia signaling pathway in Hypophthalmichthys molitrix, we cloned the phd3 gene, whose coding sequence (CDS) consists of 738 base pairs (bp) and encodes 245 amino acids. Protein structure prediction and phylogenetic tree analysis revealed that H. molitrix phd3 exhibits high evolutionary conservation. Quantitative Real-time PCR (RT-qPCR) results showed that phd3 mRNA levels were significantly upregulated under hypoxic conditions, suggesting a sensitive response of phd3 to hypoxia. Furthermore, we constructed a phd3 expression plasmid, and Western Blot analysis revealed that the protein encoded by phd3 is approximately 27 kilodaltons (kD) in size. Dual-luciferase reporter assays demonstrated that phd3significantly suppressed the activity of a hypoxia-responsive element (HRE) reporter under hypoxic conditions. To investigate the in vivo biological function of phd3, we generated phd3 mutant H. molitrix using CRISPR/Cas9 gene editing, and comparison of hypoxia tolerance between wild-type and mutant fish revealed that disruption of phd3 significantly enhanced tolerance to hypoxia. These results suggest that phd3 is significantly induced under hypoxia and negatively regulates the hypoxia signaling pathway in H. molitrix. Disrupting phd3 significantly enhances hypoxia tolerance in H. molitrix. This provides a basis for further elucidating the molecular regulatory mechanisms underlying hypoxia adaptation and offers a potential molecular target for breeding new hypoxia-tolerant strains of H. molitrix.