The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized alpha betamotif (CSab) consists of an alpha-helix and an anti-parallel triple-stranded beta-sheet connected by two pairs of disulfide bridges. The protein containing the motif share low sequence identity but high structural similarity, and it has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CSab motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty-two out of forty-six residue positions of VrD1 are altered by site-directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CSab motif. The information is valuable to the scaffold with the CSab motif and protein engineering.