Abstract: Procollagen C-proteinase enhancer protein (PCOLCE) is a secreted protein that enhances the activity of procollagen C-proteinase, thereby facilitating the maturation of collagen and influencing the process of bone formation. Down-regulation of pcolce expression compromises the maturation of collagen, ultimately leading to a reduction in bone mass. In teleost fish, the occurrence of genomic duplication events has led to the emergence of two paralogs of pcolce, designated pcolcea and pcolceb, whose functional roles in skeletal development remain incompletely characterized. To delineate the functional divergence of pcolcea and pcolceb in zebrafish (Danio rerio) osteogenesis, this study employed a multi-faceted approach integrating computational analysis of sequence features, spatiotemporal expression profiling, and phenotypic characterization of targeted mutants. The results indicated that pcolcea and pcolceb comprise 8 and 9 exons, respectively, with pcolceb being significantly longer; both genes were predominantly expressed in the vertebrae, head, and pectoral fins. Through targeted gene knockout, we generated pcolcea^-/- and pcolceb^-/- homozygous mutants. The pcolcea^-/- mutant carried a 229-bp deletion in exon 1, while the pcolceb^-/- mutant carried a 7-bp deletion in exon 3. Compared to wild-type D. rerio, pcolcea^-/- and pcolcea^-/-; pcolceb^-/- double mutant D. rerio exhibited a marked reduction in vertebral tissue mineral density, whereas pcolceb^-/- D. rerio showed no significant difference. Subsequently, the expression of genes related to bone development (runx2b, entpd5a, alpl, bglap, sp7), collagen development (col1a1a, col1a1b, col1a2), and downstream effector genes (bmp1a, bmp1b) was quantified by RT-qPCR in the caudal vertebrae of the mutants. RT-qPCR results showed that expression of bmp1b, col1a1a, col1a2, runx2b, alpl, and sp7 was significantly downregulated in pcolcea^-/- mutants. In pcolcea^-/-; pcolceb^-/- double mutants, the expression of bmp1b, col1a1a, col1a1b, col1a2, runx2b, entpd5a, bglap, and sp7 was significantly lower than in wild-type D. rerio. Collectively, these results demonstrated that loss of pcolcea impaired skeletal tissue mineralization in D. rerio, and this phenotype was significantly exacerbated by the concurrent deletion of pcolceb. This study elucidates the distinct roles of pcolcea and pcolceb in D. rerio bone development and provides a foundation for understanding the molecular mechanisms governing skeletal formation in fish.