Among vertebrates that can be kept in captivity, the annual fish Nothobranchius furzeri possesses the shortest known lifespan. It also shows typical signs of aging and is therefore an ideal model to assess the role of different physiological and environmental parameters on aging and lifespan determination. Here, we used Nothobranchius furzeri to study whether aging is associated with mitochondrial DNA (mtDNA) alterations and changes in mitochondrial function. We sequenced the complete mitochondrial genome of N. furzeri and found an extended control region. Large-scale mtDNA deletions have been frequently described to accumulate in other organisms with age, but there was no evidence for the presence of detectable age-related mtDNA deletions in N. furzeri. However, mtDNA copy number significantly decreased with age in skeletal muscle, brain, liver, skin and dorsal fin. Consistent with this finding, expression of Pgc-1α that encodes a transcriptional coactivator of mitochondrial biogenesis and expression of Tfam and mtSsbp both encoding mtDNA binding factors was downregulated with age. The investigation of possible changes in mitochondrial function revealed that the content of respiratory chain complexes III and IV was reduced in skeletal muscle with age. In addition, ADP-stimulated and succinate-dependent respiration was decreased in mitochondria of old fish. These findings suggest that despite the short lifespan, aging in N. furzeri is associated with a decline in mtDNA copy number, the downregulation of mtDNA-associated genes and an impairment of mitochondrial function.