Pepsinogen is one kind of gastric digestive proteinases belonging to a family of aspartic proteinases, which is synthesized in the gastric mucosa of vertebrates and converted to pepsins in the acidic environment of gastric juice. The mandarin fish (Siniperca chuatsi) is a typical carnivorous fish which only prey on small live full-length cDNA of pepsinogen gene of S. chuatsi) was cloned by means of RT-PCR and rapid amplification of cDNA ends (RACE) and sequenced. The result reveals a 1 367 bp cDNA full-length sequence containing 43 bp 5′-untranslated region, 187 bp 3′-untranslated region and 1 137 bp open reading frame (ORF), which encodes 378 amino acids with characteristic signal peptides and activation peptides. The pepsinogen of S. chuatsi also has highly conserved amino acid residues essential for catalytic activity and conformational maintenance, which are two essential aspartyl residues in the active site and six cysteine residues involved in forming three disulphide bonds. The homology of amino acid sequences between pepsinogen of S. chuatsi and other vertebrate pepsinogens ranges from 59.9% to 91.2%, which indicates the pepsinogen gene is highly conserved in evolution. The NJ phylogenetic tree of vertebrates from the amino acids sequences of pepsinogen showed all fish were clustered as a group, the pepsinogen of S. chuatsi was the closest to pepsinogen A form Ⅱ a of winter flounder. The successful cloning of pepsinogen gene from S. chuatsi not only lays the foundation for further study of its temporal and spatial expression, but also provides new material for the molecular characteristic and evolution of fish pepsinogen.