Arabidopsis Clade 3 GLUTAMATE RECEPTOR-LIKEs (GLRs) are primary players in wound-induced systemic signaling. Previous studies focused on dissecting their ligand-activated channel properties involving extracellular and membrane-related domains. Here, we report that the carboxy-terminal tails (C-tails) of GLRs contain key elements controlling their function in wound signaling. GLR3.3 without its C-tail failed to rescue the glr3.3a mutant. We carried out a yeast two-hybrid screen to identify the C-tail interactors. We performed functional studies of the interactor by measuring electrical signals and defense responses. Then we mapped their binding sites and evaluated the impact of the sites on GLR functions. IMPAIRED SUCROSE INDUCTION 1 (ISI1) interacted with GLR3.3. Enhanced electrical activity was detected in reduced function isi1 mutants in a GLR3.3-dependent manner. isi1 mutants were slightly more resistant to insect feeding than the wild-type. Furthermore, a triresidue motif RFL in the GLR3.3 C-tail binds to ISI1 in yeast. Finally, we demonstrated that FL residues were conserved across GLRs and functionally required. Our study provides new insights into the functions of GLR C-tails, reveals parallels with the ionotropic glutamate receptor regulation in animal cells, and may enable rational design of strategies to engineer GLRs for future practical applications.