Type 1 ryanodine receptor (RyR1) is a Ca²⁺ release channel in the sarcoplasmic reticulum of skeletal muscle and plays an important role in excitation-contraction coupling. Genetic mutations in the RyR1 gene accelerate the channel activity to cause several muscle diseases including malignant hyperthermia (MH). Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We have recently developed a novel RyR1-selective inhibitor, Compound 1, a derivative of oxolinic acid with better water solubility and short plasma half-life. To understand molecular mechanism of RyR1-selective inhibition by Compound 1, we carried out identification of binding site for the compound on RyR1. Binding domains were searched by various chimeric channels of RyR1 and RyR2 using ER Ca²⁺ measurements with R-CEPIA1er, a fluorescent ER Ca²⁺ indicator. We successfully narrowed down the binding site within a domain comprising ~200 amino acid residues. Coordination of Compound 1 was predicted by fitting the compound on high-resolution cryo-EM structure of RyR1 and was validated by mutagenesis of residues in the binding pocket. Our data will provide rational design for more potent and selective inhibitors for treatment of RyR1-related diseases.