Cystatins, the natural inhibitors of cysteine proteases are important regulatory proteins found in mammals, plants, and insects. Plant cystatins (phytocystatins) show high degree of homology with the members in animal cystatin family. Phytocystatins have several possible functions, including regulating the activity of endogenous cysteine proteases during different physiological processes such as seed maturation and germination, as well as responding to biotic and abiotic stresses. They may also be involved in defense mechanisms to protect plants from the invasion of pathogens or the attack by pests. Phytocystatins appear to function as inhibitors of both endogenous and exogenous cysteine proteases and therefore may have potential applications in agriculture and medicine. We have successfully cloned a phytocystatin gene from seasame seed (Sesamum indicum L.). According to the phytogenetic analysis, the recombinant seasame cystatin (SiCYS, 199 residues) possess an N-terminal cystatin domain (SiCYSN), and a C-terminal cystatin-like domain (SiCYSC). In order to understand the structure and function of SiCYS, we have used a fluorescence activity assay and multi-dimensional NMR to characterize the recombinant SiCYS. The recombinant full length SiCYS showed inhibitory activity toward a cysteine protease, papain with inhibitory constant, KI of 1 x 10-8 M. SiCYSN also showed strong inhibitory activity toward papain with KI of 4 x 10-9 M, however, SiCYSC showed much weaker inhibitory activity toward papain with KI of 2 x 10-6 M. The NMR structure showed that SiCYSN contained a compact inhibitory domain comprising a four-stranded antiparallel β-sheet wrapped around a central α-helix. The three structural motifs (G5, Q46XVXG, and W77) putatively responsible for the interaction with papain-like proteases are located in one side. SiCYSC also showed a cystatin-like structure, with four-stranded antiparallel β-sheet and a central α-helix with only 30% sequence identity to SiCYSN. However, SiCYSC contained shorter secondary structures and a much less stable core structure.