Ankyrin repeat and suppression-of-cytokine-signaling box (Asb) proteins, a subset of ubiquitin ligase E3, include Asb5 with six ankyrin-repeat domains. Zebrafish harbor two gene isoforms, and . Currently, the effects of gene inactivation on zebrafish embryonic development and heart function are unknown. Using CRISPR/Cas9, we generated -knockout zebrafish, revealing no abnormal phenotypes at 48 h post-fertilization (hpf). In situ hybridization showed similar and expression patterns, indicating the functional redundancy of these isoforms. Morpholino interference was used to target in wild-type and -knockout zebrafish. Knocking down in the wild-type had no phenotypic impact, but simultaneous knockdown in -knockout homozygotes led to severe pericardial cavity enlargement and atrial dilation. RNA-seq and cluster analyses identified significantly enriched cardiac muscle contraction genes in the double-knockout at 48 hpf. Moreover, semi-automatic heartbeat analysis demonstrated significant changes in various heart function indicators. STRING database/Cytoscape analyses confirmed that 11 cardiac-contraction-related hub genes exhibited disrupted expression, with three modules containing these genes potentially regulating cardiac contractile function through calcium ion channels. This study reveals functional redundancy in and , with simultaneous knockout significantly impacting zebrafish early heart development and contraction, providing key insights into 's mechanism.