Ca2+ ions have distinct roles in the outer segment, cell body, and synaptic terminal of photoreceptors. We tested the hypothesis that distinct Ca2+ domains are maintained by Ca2+ uptake into mitochondria. Serial block face scanning electron microscopy of zebrafish cones revealed that nearly 100 mitochondria cluster at the apical side of the inner segment, directly below the outer segment. The endoplasmic reticulum surrounds the basal and lateral surfaces of this cluster, but does not reach the apical surface or penetrate into the cluster. Using genetically encoded Ca2+ sensors, we found that mitochondria take up Ca2+ when it accumulates either in the cone cell body or outer segment. Blocking mitochondrial Ca2+ uniporter activity compromises the ability of mitochondria to maintain distinct Ca2+ domains. Together, our findings indicate that mitochondria can modulate subcellular functional specialization in photoreceptors.SIGNIFICANCE STATEMENT Ca2+ homeostasis is essential for the survival and function of retinal photoreceptors. Separate pools of Ca2+ regulate phototransduction in the outer segment, metabolism in the cell body, and neurotransmitter release at the synaptic terminal. We investigated the role of mitochondria in compartmentalization of Ca2+ We found that mitochondria form a dense cluster that acts as a diffusion barrier between the outer segment and cell body. The cluster is surprisingly only partially surrounded by the endoplasmic reticulum, a key mediator of mitochondrial Ca2+ uptake. Blocking the uptake of Ca2+ by mitochondria causes redistribution of Ca2+ throughout the cell. Our results show that mitochondrial Ca2+ uptake in photoreceptors is complex and plays an essential role in normal function.