Bestrophin-1 (Best1) is a calcium (Ca²⁺)-activated chloride (Cl^-) channel which has a phylogenetically conserved channel structure with an aperture and neck in the ion-conducting pathway. Mammalian mouse Best1 (mBest1) has been known to have a permeability for large organic anions including gluconate, glutamate, and D-serine, in addition to several small monovalent anions, such as Cl^‑, bromine (Br^-), iodine (I^-), and thiocyanate (SCN^-). However, it is still unclear whether non-mammalian Best1 has a glutamate permeability through the ion-conducting pathway. Here, we report that chicken Best1 (cBest1) is permeable to glutamate in a Ca²⁺-dependent manner. The molecular docking and molecular dynamics simulation showed a glutamate binding at the aperture and neck of cBest1 and a glutamate permeation through the ion-conducting pore, respectively. Moreover, through electrophysiological recordings, we calculated the permeability ratio of glutamate to Cl^- (P_Glutamate/P_Cl) as 0.28 based on the reversal potential shift by ion substitution from Cl^- to glutamate in the internal solution. Finally, we directly detected the Ca²⁺-dependent glutamate release through cBest1 using the ultrasensitive two-cell sniffer patch technique. Our results propose that Best1 homologs from non-mammalian (cBest1) to mammalian (mBest1) have a conserved permeability for glutamate.