The function of zebrafish transcription factor hoxb4a in the heart has not been reported. In order to clarify the role of hoxb4a in heart development, gene silencing and mRNA overexpression were used to knock down and overexpress hoxb4a gene in zebrafish, respectively. It was found that the embryo with inhibition or overexpression of hoxb4a had the phenotype of pericardial enlargement and abnormal cardiac looping at 3 days poster fertilization. Compared with wild-type and hoxb4a knockdown embryos by in situ hybridization, the measurement results showed that the heart looping angle of hoxb4a knockdown embryos increased. Then, using transgenic fish lines, we found abnormal endocardial abnormalities of pharyngeal arch in embryos after knockdown of hoxb4a. Furthermore, qPCR was used to detect the expression changes of heart-related genes after hoxb4a silencing. The results showed that most of the key genes of heart development were significantly down-regulated after hoxb4a knockdown, while the key gene nppa regulating cardiac differentiation was significantly up-regulated. This study shows that silencing of hoxb4a in zebrafish can lead to abnormal cardiac structure and looping, and regulate the changes of early cardiac development genes. In this study, we found that hoxb4a plays a role in the early cardiac development of zebrafish, and preliminarily explored the regulation of hoxb4a on the differentiation of cardiac chambers and cardiac looping through in situ hybridization and qPCR results, which deepened the understanding of the regulatory network of cardiac development.