Disulfide bonds play a crucial role in the folding and structural stabilization of many important extracellular peptides and proteins including hormones, enzymes, growth factors, toxins, and immunoglobulins. Cysteine-rich peptides stabilized by intramolecular disulfide bonds have often been isolated from venoms of microbes, animals and plants. These peptides typically have much higher stability and improved biopharmaceutical properties compared to their linear counterparts. Therefore the correct disulfide bond formation of small proteins and peptides has been extensively studied for a better understanding of their folding mechanism and achieving efficient generation of the naturally occurring biologically active product. Imperatoxin A (IpTxa), a peptide toxin containing 6 cysteine residues, was isolated from the venom of scorpion Pandinus imperator, selectively binds the ryanodine receptors and activates Ca release from sarcoplasmic reticulum (SR). IpTxa increases the binding of ryanodine to ryanodine receptors (RyRs) and encourages reconstituted single channel to induce subconductance states. We previously reported the three-dimensional structure of IpTxa determined by solution NMR spectroscopy. 6 The molecular structure of IpTxa consists of two antiparallel β-strands connected by four chain reversals. The overall structure of IpTxa is stabilized by three disulfide bonds. This topology and disulfide bond pattern is classified as an ‘inhibitor cysteine knot’ fold found in numerous toxic and inhibitory peptides, including ω-conotoxins. Figure 1 shows the sequence alignment of IpTxa with ω-conotoxin GVIA, MVIIA, and MVIIC demonstrating the same number and position of cysteines, although they have very low Dali Z-scores and sequence identity. IpTxa exhibits a large functional surface area identified by alanine-scanning mutagenesis, and the hydrophobic core of IpTxa is entirely composed of the four out of six cysteines that form intramolecular disulfide bonds to stabilize the overall toxin structure. Without cysteine residues, IpTxa completely lost its molecular structure and biological activity. These results indicate that the information of the authentic disulfide bond connectivity of cysteine-rich peptides or proteins might help not only to understand their three-dimensional conformation but to prepare biologically active products. Here we report the disulfide bond connectivity of IpTxa determined by a combined approach of enzymatic fragmentation and chemical synthesis. The disulfide bond connectivity of IpTxa is the same as that of ω-conotoxins (between the 1 and 4, 2 and 5, and 3 and 6 cysteines). The role of disulfide bonds of IpTxa is discussed.