Neurons of the retina require oxygen to survive. In hypoxia, neuronal ATP production is impaired, ATP-dependent ion pumping is reduced, transmembrane ion gradients are dysregulated, and intracellular Ca²⁺ concentration ([Ca²⁺]_i) increases enough to trigger excitotoxic cell death. Central neurons of the common goldfish (Carassius auratus) are hypoxia tolerant, but little is known about how goldfish retinas withstand hypoxia. To study the cellular mechanisms of hypoxia tolerance, we isolated retinal interneurons (horizontal cells; HCs), and measured [Ca²⁺]_i with Fura-2. Goldfish HCs maintained [Ca²⁺]_i throughout 1 h of hypoxia, whereas [Ca²⁺]_i increased irreversibly in HCs of the hypoxia-sensitive rainbow trout (Oncorhynchus mykiss) with just 20 min of hypoxia. Our results suggest mitochondrial ATP-dependent K⁺ channels (mK_ATP) are necessary to stabilize [Ca²⁺]_i throughout hypoxia. In goldfish HCs, [Ca²⁺]_i increased when mK_ATP channels were blocked with glibenclamide or 5-hydroxydecanoic acid, whereas the mK_ATP channel agonist diazoxide prevented [Ca²⁺]_i from increasing in hypoxia in trout HCs. We found that hypoxia protects against increases in [Ca²⁺]_i in goldfish HCs via mK_ATP channels. Glycolytic inhibition with 2-deoxyglucose increased [Ca²⁺]_i, which was rescued by hypoxia in a mK_ATP channel-dependent manner. We found no evidence of plasmalemmal K_ATP channels in patch-clamp experiments. Instead, we confirmed the involvement of K_ATP in mitochondria with TMRE imaging, as hypoxia rapidly (<5 min) depolarized mitochondria in a mK_ATP channel-sensitive manner. We conclude that mK_ATP channels initiate a neuroprotective pathway in goldfish HCs to maintain [Ca²⁺]_i and avoid excitotoxicity in hypoxia. This model provides novel insight into the cellular mechanisms of hypoxia tolerance in the retina.