Transfer ribonucleic acid (tRNA) is the RNA molecule with the largest variety of post-transcriptional modifications. In particular its anticodon loop contains a large number of modifications. Mitochondria have a relatively independent protein synthesis system, mitochondrial tRNAs (mt-tRNAs) are all encoded by the mitochondrial genome. Studies have shown that 5-taurinomethyluridine modification (τm⁵U) only exists at position 34 of mitochondrial tRNAs of higher eukaryotes, regulating the fidelity of codon and anticodon interaction and contributing to translation speed and fidelity. Human GTPBP3 and MTO1 mediate mitochondrial τm⁵U modification, whose functional defects may cause mitochondrial encephalomyopathy. This review summarizes the biological properties of τm⁵U modification and its modifying enzymes, providing a new insight into the mechanism of τm⁵U modification and the pathogenesis of mitochondrial diseases caused by τm⁵U modification defects.