Imperatoxin A (IpTxa) is known to modify the gating of skeletal ryanodine receptor (RyR1). In this paper, the ability of charged aa residues of IpTxa to induce substate of native RyR1 in HSR was examined. Our results show that the basic residues (e.g., Lys19, Lys20, Lys22, Arg23, and Arg24) are important for producing substate of RyR1. In addition, other basic residues (e.g., Lys30, Arg31, and Arg33) near the C‐terminus and some acidic residues (e.g., Glu29, Asp13, and Asp2) are also involved in the generation of substate. Residues such as Lys8 and Thr26 may be involved in the self‐regulation of substate of RyR1, since alanine substitution of the aa residues led to a drastic conversion to the substate. The modifications of the channel gating by the wild‐type and mutant toxins were similar in purified RyR1. Taken together, the specific charge distributions on the surface of IpTxa are essential for regulation of the channel gating of RyR1.