【】: Objective To characterize the expression pattern of transcription factor tfe3b and investigate its role and molecular mechanism in regulating cardiac morphogenesis in zebrafish. Methods A tfe3b knockout line was generated using CRISPR/Cas9 technology targeting exon 1. Spatiotemporal expression of tfe3b and cardiac markers (cmlc2, tnnt2a, etc.) was detected via whole-mount in situ hybridization (WISH). Phenotypic defects were quantified in embryos from 30 to 96 hpf (n>30 per group). Transcriptome sequencing (RNA-seq) followed by Gene Ontology (GO) enrichment analysis was performed on heart tissues isolated at 48 hpf, with key differentially expressed genes (DEGs) validated by qPCR. Results tfe3b was specifically expressed in the zebrafish heart from 36 to 48 hpf. tfe3b?/? mutants exhibited progressive pericardial edema starting at 54 hpf. Phenotypic identification revealed that while cardiac jogging at 30 hpf was unaffected, cardiac looping was significantly impaired at 48 hpf, characterized by reduced curvature and mesocardia. RNA-seq analysis identified significant downregulation of genes associated with cardiomyocyte differentiation, fatty acid oxidation, and the tricarboxylic acid (TCA) cycle in mutants. Conclusion tfe3b is a novel and specific regulator of cardiac looping rather than early asymmetric migration. Its deficiency leads to impaired morphogenesis by perturbing the coordination between energy metabolism and myocardial development, providing new genetic insights into vertebrate cardiogenesis.