Mitochondrial transport is a complex process involving different pathways for protein localization and each of the four gaps of mitochondria (OMM, IMM, IMS, and mitochondrial matrices). More than 1,300 protein molecules in mammalian mitochondria, only 13 are encoded by mtDNA, and the rest are encoded by nDNA, which are first synthesized in the cytoplasm and then transported to specific parts of the mitochondria. Therefore, these mitochondria proteins require a specific protein transport machine is used to complete the process. The transport machine is designed for mitochondrial input and assembly pathways of IMS proteins. It is consist with the outer membrane translocation enzyme (TOM) and endomembrane translocase (TIM), sorting and assembly machinery (SAM), and the leader sequence shift enzyme-related motor (PAM). Among them, TOM and TIM run new peptides into the corresponding gaps. Therefore, some mutations in the peptides are associated with specific enzyme defects, such as methylmalonate and pyruvate dehydrogenase complex (PDHC) deficiency. Clinical studies find some human diseases are caused by the total transport mechanism. For example, one is deafness/dystonia peptide 1 (DDP1) mutation leading to an X-linked recessive hereditary deafness, dystonia syndrome, and the other is a chaperone HSP60 mutation leading to an autosomal dominant hereditary hernia sexual paraplegia (HSP).