Our previous study generated a series of cybrids containing mitochondria of synaptosomes from mice at different ages. The following functional analysis on these cybrids revealed an age-dependent decline of mitochondrial function. To understand the underlying mechanisms that contribute to the age-related mitochondrial dysfunction, we focused on three cybrids carrying mitochondria derived from synaptosomes of the old mice that exhibited severe respiratory deficiencies. In particular, we started with a comprehensive analysis of mitochondrial genome by high resolution, high sensitive deep sequencing method. Compared with young, control, we detected a significant accumulation of heteroplasmic mtDNA mutations. These mutations included six alterations in main control region that has been shown to regulate overall gene-expression, and four alterations in protein coding region, two of which led to significant changes in complex I subunit ND5 and complex III subunit CytB. Interestingly, a reduced mtDNA-encoded protein synthesis was associated with the changes in the main control region. Likewise, mutations in ND5 and CytB were associated with defects in assembly of respiratory complexes. Altogether, the identified age-dependent accumulation of mtDNA mutations in mouse brain likely contributes to the decline in mitochondrial function.