Objective To establish Prpf40b knockout rats for biological research of the gene and explore the effect of gene deletion on development of the heart. Methods Prpf40b knockout rats were established by CRISPR/ Cas9 technology. The genotypes of the founder rats and offspring were identified by sequencing and PCR. The cardiac structure and function of knockout rats were analyzed by ultrasound imaging technology. The microscopic morphology of the rat myocardium was analyzed by histopathological observation. Results PCR and sequencing confirmed successful establishment of Prpf40b gene knockout rats. Compared with the wt rats, ultrasound imaging analysis showed that the cardiac structure and morphology of 2-month-old knockout rats were not significantly abnormal; whereas the ventricular cavity diameter and volume of 12-month-old knockout rats decreased significantly in the systole and diastole, and the ejection fraction decreased significantly. Histopathological analysis showed that the myocardium of 12-month-old knockout rats was arranged irregularly, the thickness of myocardial fibers was uneven, and the sarcoplasmic reticulum was dilated. Conclusions Deletion of the Prpf40b gene induces changes in the overall structure and morphology of the rat heart and abnormal myocardial histology.