Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function as well as susceptibility to various diseases such as Parkinson disease, Alzheimer disease, bipolar disorder, and cancer. However, it is unclear whether and how mtDNA polymorphisms affect intracellular function, such as calcium signaling or pH regulation. Here we searched for mtDNA polymorphisms that have intracellular functional significance using transmitochondrial hybrid cells (cybrids) carrying ratiometric Pericam (RP), a fluorescent calcium indicator, targeted to the mitochondria and nucleus. By analyzing the entire mtDNA sequence in 35 cybrid lines, we found that two closely linked nonsynonymous polymorphisms, 8701A and 10398A, increased the basal fluorescence ratio of mitochondria-targeted RP. Mitochondrial matrix pH was lower in the cybrids with 8701A/10398A than it was in those with 8701G/10398G, suggesting that the difference observed by RP was mainly caused by alterations in mitochondrial calcium levels. Cytosolic calcium response to histamine also tended to be higher in the cybrids with 8701A/10398A. It has previously been reported that 10398A is associated with an increased risk of Parkinson disease, Alzheimer disease, bipolar disorder, and cancer, whereas 10398G associates with longevity. Our findings suggest that these mtDNA polymorphisms may play a role in the pathophysiology of these complex diseases by affecting mitochondrial matrix pH and intracellular calcium dynamics.