BACKGROUND: Conopeptides are neuropharmacological peptides derived from the venomous salivary glands of cone snails. Among 29 superfamilies based on conserved signal sequences, T-superfamily conotoxins, which belong to the smallest group, include four different frameworks that contain four cysteines denominated I, V, X and XVI. In this work, the primary structure and the cysteine connectivity of novel conotoxin of Conus bandanus were determined by tandem mass spectrometry using collision-induced dissociation. METHODS: The venom glands of C. bandanus snails were dissected, pooled, and extracted with 0.1% trifluoroacetic acid in three steps and lyophilized. The venom was fractionated and purified in an HPLC system with an analytical reversed-phase C18 column. The primary peptide structure was analyzed by MALDI TOF MS/MS using collision-induced dissociation and confirmed by Edman's degradation. The peptide's cysteine connectivity was determined by rapid partial reduction-alkylation technique. RESULTS: The novel conotoxin, NGC1C2(I/L)VREC3C4, was firstly derived from de novo sequencing by MS/MS. The presence of isoleucine residues in this conotoxin was confirmed by the Edman degradation method. The conotoxin, denominated Bn5a, belongs to the T1-subfamily of conotoxins. However, the disulfide bonds (C1-C4/C2-C3) of Bn5a were not the same as found in other T1-subfamily conopeptides but shared common connectivities with T2-subfamily conotoxins. The T1-conotoxin of C. bandanus proved the complexity of the disulfide bond pattern of conopeptides. The homological analysis revealed that the novel conotoxin could serve as a valuable probe compound for the human-nervous-system norepinephrine transporter. CONCLUSION: We identified the first T1-conotoxin, denominated Bn5a, isolated from C. bandanus venom. However, Bn5a conotoxin exhibited unique C1-C4/C2-C3 disulfide connectivity, unlike other T1-conotoxins (C1-C3/C2-C4). The structural and homological analyses herein have evidenced novel conotoxin Bn5a that may require further investigation.