Canonical-type transient receptor potential cation channel type 3 (TRPC3) allows the entry of extracellular Ca2+ and Na+ into various cells. In mouse skeletal myotubes, functional interaction between TRPC3 and RyR1 (ryanodine receptor type 1/Ca2+-release channel on sarcoplasmic reticulum membrane) regulates the gain of excitation–contraction coupling. Junctophilin-2 (JP2) is a TRPC3-interacting protein in mouse skeletal myotubes. Based on these knowledge from bona-fide TRPC3-expressing cells, to identify critical binding region(s) of JP2 that participate in binding to TRPC3, various JP2 portions were subjected to co-immunoprecipitation assay with intact TRPC3 from rabbit skeletal muscle. A region covering 143 to 234 amino acids of JP2 (F1-2) was the most efficient portion binding to TRPC3. Through mutational studies, we found that the binding ability of JP2 to TRPC3 was mainly due to glutamate in the F1-2 region (E227). This substantial binding between JP2 and TRPC3 suggests that JP2 can be a regulatory protein of TRPC3 and/or TRPC3-mediated Ca2+ homeostasis in skeletal muscle.