The zebrafish model has been widely used to investigate numerous signaling pathways in vertebrates, including programmed cell death. Although several zebrafish proteins homologous to mammalian caspases have been identified, our understanding of these zebrafish caspases is still limited. Recently, we identified a large number of natural caspase-3 substrates from the human proteome by using the mRNA-display selection method. Through comparative analysis, we found that the cleavage sites on some of these novel human caspase-3 substrates are highly conserved in their zebrafish orthologs. We report here the identification and characterization of 14 natural zebrafish caspase-3 substrates that have not yet been previously studied. The specific cleavage of these zebrafish proteins was compared with caspases from different species, and the protein fragments that contain the putative cleavage sites were mapped. The work described here could facilitate our understanding of the downstream signaling pathways that are mediated by caspase-3 in zebrafish.