To function properly, mitochondria utilize products of 37 and >1,000 genes encoded by the mitochondrial and nuclear genomes, respectively, which should be compatible with each other. Discordance between mitochondrial and nuclear genetic ancestry could contribute to phenotypic variation in admixed populations. Here we explored potential mito-nuclear incompatibility in six admixed human populations from the Americas: African Americans, African Caribbeans, Colombians, Mexicans, Peruvians, and Puerto Ricans. For individuals in these populations, we determined nuclear genome proportions derived from Africans, Europeans, and Native Americans, the geographic origins of the mitochondrial DNA (mtDNA), as well as mtDNA copy number in lymphoblastoid cell lines. By comparing nuclear vs. mitochondrial ancestry in admixed populations, we show that, first, mtDNA copy number decreases with increasing discordance between nuclear and mitochondrial DNA ancestry, in agreement with mito-nuclear incompatibility. The direction of this effect is consistent across mtDNA haplogroups of different geographic origins. This observation suggests suboptimal regulation of mtDNA replication when its components are encoded by nuclear and mtDNA genes with different ancestry. Second, while most populations analyzed exhibit no such trend, in Puerto Ricans and African Americans we find a significant enrichment of ancestry at nuclear-encoded mitochondrial genes towards the source populations contributing the most prevalent mtDNA haplogroups (Native American and African, respectively). This likely reflects compensatory effects of selection in recovering mito-nuclear interactions optimized in the source populations. Our results provide the first evidence of mito-nuclear effects in human admixed populations and we discuss its implications for human health and disease.