Photoreceptor ribbon synapses tonically release glutamate. To ensure efficient signal transmission and prevent glutamate toxicity, a highly efficient glutamate removal system provided by members of the SLC1 gene family is required. By using a combination of biophysical and in vivo studies, we elucidate the role of excitatory amino acid transporter 2 (EAAT2) proteins in synaptic glutamate homeostasis at the zebrafish photoreceptor synapse. The main glutamate sink is provided by the glial EAAT2a, reflected by reduced electroretinographic responses in EAAT2a-depleted larvae. EAAT2b is located on the tips of cone pedicles and contributes little to glutamate reuptake. However, this transporter displays both a large chloride conductance and leak current, being important in stabilizing the cone resting potential. This work demonstrates not only how proteins originating from the same gene family can complement each other's expression profiles and biophysical properties, but also how presynaptic and glial transporters are coordinated to ensure efficient synaptic transmission at glutamatergic synapses of the central nervous system.