Protein kinase D1 (PKD1 or PRKD1) is a member of the protein kinase family, which is composed of three structurally related stress-activating enzymes that regulate a variety of biological functions in the body, mainly involving cell proliferation, differentiation, apoptosis, immune regulation, cardiac contraction, angiogenesis, and cancer. In these processes, PRKD1 is associated with substrate phosphorylation of cardiac hypertrophy, systole, and ischemia-reperfusion injury. Relevant studies have reported the presence of PRKD1 gene mutations in patients with congenital heart disease, but its specific function and molecular mechanism in the heart have not been elucidated. In order to facilitate the study of the mechanism of PRKD1 gene in early human heart development, a zebrafish prkd1 gene knockout line was constructed using CRISPR/Cas9 technology. Firstly, the two optimal gene knockout target sites were screened out through the bioinformatics website, and the single guide RNAs (sgRNAs) and primers of the corresponding target sites were synthesized. Then, the sgRNAs of the two target sites were transcribed in vitro and mixed with Cas9 protein and co-in-jected into the one-cell stage zebrafish embryos. Finally, the zebrafish embryos and adults of the F0, F1, F2 and F3 generations after gene knockout were identified and phenotypically observed. The results showed that there were different base deletions near the target site. Three sublineages of prkd1 gene knockout that can be stably inherited in the F1 generation were successfully constructed. Compared with the wild type, the F3 homozygous embryos showed different degrees of malformations such as cardiac chamber expansion, abnor-mal circularization and cardiac tube linearization. In summary, this study demonstrated the successful con-struction of a zebrafish prkd1 gene knockout line using CRISPR/Cas9 technology, which lays a groundwork for further studying the specific function of this gene in human heart development and is useful for congeni-tal heart disease screening and precision medicine.