Cryptochrome 4a (Cry4a) is a magnetically sensitive protein thatcould enable night-migratory birds to sense the geomagnetic field for navigation. The key to the protein's magnetic sensitivity is the flavin adenine dinucleotide (FAD) cofactor, which initiates the electron transfer within the protein, leading to a spincorrelated radical pair. Despite its importance, the mechanism of FAD binding in avian Cry4a proteins remains unclear. Here, we show that a point mutation of the positively charged arginine residue at position 356 to a negatively charged glutamic acid (R356E) completely depletes FAD binding from European robin (Erithacus rubecula) Cry4a. The result indicates that electrostatic interactions constitute the primary driving force that enables the FAD binding in European robin Cry4a. The finding provides new structural insight into the molecular basis of FAD binding in Cry4 and advances our understanding of the biophysical underpinnings of bird magnetoreception.