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Jingzhaotoxin-Ⅰ (JZTX-Ⅰ) purified from the venom of the spider Chilobrachys jingzhao is a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3.The structure of this toxin in aqueous solution was investigated using 2-D 1H-NMR techniques. The complete sequence-specific assignments of proton resonance in the 1H-NMR spectra of JZTX-Ⅰ were obtained by analyzing a series of 2-D spectra, including DQF-COSY, TOCSY and NOESY spectra, in H2O and D2O. All the backbone protons except for Gln4 and more than 95% of the side-chain protons were identified by dαN,dαδ, dβN and dNN connectivities in the NOESY spectrum. These studies provide a basis for the further determination of the solution conformation of JZTX-Ⅰ. Furthermore, the secondary structure of JZTX-Ⅰ was identified from NMR data. It consists mainly of a short triple-stranded antiparallel β-sheet with Trp7-Cys9, Phe20-Lys23 and Leu28-Trp31. The characteristics of the secondary structure of JZTX-Ⅰ are similar to those of huwentoxin-Ⅰ (HWTX-Ⅰ) and hainantoxin-Ⅳ (HNTX-Ⅳ), whose structures in solution have previously been reported.