In this paper, we report the molecular cloning of a novel stefin analogue from the spleen of large yellow croaker Pseudosciaena crocea (Lycstefin).The open reading frame (ORF) of 297 nucleotides (nt) of Lycstefin encodes a protein of 99 amino acids (aa) with a putative molecular weight of 1 l kDa, in which no signal peptide and potential N-glycoslation site are predicted.The deduced Lycstefin possesses the structural features of the mammalian stefins, including two conserved motifs known to interact with the active sites of family C 1 cysteine peptidases: one glycine in the N-terminal region (G6) and GIn-Xaa-Val-Xaa-Gly motif (Q48LVAG52).It shares 32-47.5% aa sequence identity to the sequences found in mammals and other fish species and is rich in cysteine residues (seven cysteines).