Human mitochondrial DNA (mtDNA) variants and haplogroups may contribute to susceptibility to various diseases and pathological conditions, but the underlying mechanisms are not well understood. To address this issue, we established a cytoplasmic hybrid (cybrid) system to investigate the role of mtDNA haplogroups in human disease; specifically, we examined the effects of East Asian mtDNA genetic backgrounds on oxidative phosphorylation (OxPhos). We found that mtDNA single nucleotide polymorphisms such as m.489T>C, m.10398A>G, m.10400C>T, m.C16223T, and m.T16362C affected mitochondrial function at the level of mtDNA, mtRNA, or the OxPhos complex. Macrohaplogroup M exhibited higher respiratory activity than haplogroup N owing to its higher mtDNA content, mtRNA transcript levels, and complex III abundance. Additionally, haplogroup M had higher reactive oxygen species levels and NAD+/NADH ratios than haplogroup N, suggesting difference in mitonuclear interactions. Notably, subhaplogroups G2, B4, and F1 appeared to contribute significantly to the differences between haplogroups M and N. Thus, our cybrid-based system can provide insight into the mechanistic basis for the role of mtDNA haplogroups in human diseases and the effect of mtDNA variants on mitochondrial OxPhos function. In addition, studies of mitonuclear interaction using this system can reveal predisposition to certain diseases conferred by variations in mtDNA.