Abstract Pancreatic ribonuclease 10，which is encoded by RNase10, is a vertebrate-specific reproductive enzyme. Studies of RNase10 mainly focus on physiological function, while molecular evolution is not commonly addressed. Here, 26 RNase10s from 24 available genomic sequences of Cetartiodactyla species were identified. Surprisingly, gene duplications were mostly detected in the Artiodactyla species of Bubalus bubalis and Sus scrofa, whereas only a single RNase10 was observed in the other 22 species examined. Phylogenetic analyses with diverse methods resulted in a well-resolved phylogeny, dividing Cetacea into Odontoceti and Mysticeti, supporting monophyly of Tylopoda and Suina. The signature motif CKXXNTF has been changed and the isoelectric point (pI) is lower than that for any other RNASE A members, raising the possibility that Cetartiodactyla RNase10 may lack ribonuclease or antibacterial activities. Moreover, 13 positive sites were detected. Two of the 13 sites are observed to near the structure Cysteine of the RNase10 enzyme. Our results demonstrate the evolutionary mechanism and establishe an important theoretical foundation for future functional assays in Cetartiodactyla RNase10.