Conotoxins are well known for their highly variable structures and functions. Here we report the identification of a novel conotoxin named mrle from Conus marmoreus.mr1e is composed of 11 amino acid residues cross-linked by two disulfide bonds (CCHSSWCKHLC). The spacing of intercysteine loops in mrle is exactly the same as that in α4/3 conotoxins. However, the native mr1e peptide coeluted on reverse-phase HPLC with the regioselectively synthesized ribbon disulfide linkage isomer (C1=C4, C2-C3)but not the globular linkage isomer (C1-C3, C2-C4). Although this peptide has the same disulfide connectivity as the X-conotoxins, their sequences do not share significant homology. Thus, mrle could be defined as a novel conotoxin family. By intracranial injection into mice, mr1e showed an excitatory effect. The characterization of mr1e certainly enriches our understanding of conotoxins, and also opens an avenue for further structural and functional investigation.