Abstract: In order to investigate the role of the tent5c/fam46c gene in the development of intermuclar bones in zebrafish (Danio rerio), we analyzed the evolutionary characteristics of the tent5c gene across 11 fish species. Spatiotemporal expression patterns of tent5c gene during the zebrafish development were analyzed using in situ hybridization (ISH) and qRT-PCR. Homozygous tent5c^–/– mutant lines were established via CRISPR/Cas9 to observe skeletal phenotype and analyze gene expression changes. Analysis of the amino sequence revealed high conservation of the tent5c gene among 11 different fish species, all sharing a common structural domain. The ISH results showed that tent5c exhibited RNA level expression in the eyes and vertebrae of zebrafish at 48hpf (hours post fertilization), with particularly high expression in intermusclar bones and vertebrae at 60dpf (days post fertilization). Bone staining of tent5c^+/+ wild type zebrafish and tent5c^–/– strain showed that loss of the tent5c gene resulted in a reduced growth of intermuscular bones, decreased mineralization, and a slight decline in numbers. Transcriptome analysis revealed that tent5c knockout down-regulated 112 genes and up-regulated 118 genes. KEGG enrichment analysis indicated enrichment of differently expressed genes (DEGs) mainly in motor proteins, ECM-receptor interaction, VEGF signaling pathway, and p53 signaling pathway. These findings indicated that tent5c may influence the development of intermuscular bones through pathways related to muscle and bone. Overall, this study provides preliminary evidence of the role of the tent5c gene in zebrafish intermuscular bone development, laying a scientific foundation for further exploration of the molecular regulatory mechanisms underlying fish intermusclar bone and skeletal development.