Modified uridine containing taurine, 5-taurinomethyluridine (τm 5U), is found at the anticodon first position of mitochondrial (mt-)transfer RNAs (tRNAs). Previously, we reported that τm 5U is absent in mt-tRNAs with pathogenic mutations associated with mitochondrial diseases. However, biogenesis and physiological role of τm 5U remained elusive. Here, we elucidated τm 5U biogenesis by confirming that 5,10-methylene-tetrahydrofolate and taurine are metabolic substrates for τm 5U formation catalyzed by MTO1 and GTPBP3. GTPBP3-knockout cells exhibited respiratory defects and reduced mitochondrial translation. Very little τm 5U34 was detected in patient’s cells with the GTPBP3 mutation, demonstrating that lack of τm 5U results in pathological consequences. Taurine starvation resulted in downregulation of τm 5U frequency in cultured cells and animal tissues (cat liver and flatfish). Strikingly, 5-carboxymethylaminomethyluridine (cmnm 5U), in which the taurine moiety of τm 5U is replaced with glycine, was detected in mt-tRNAs from taurine-depleted cells. These results indicate that tRNA modifications are dynamically regulated via sensing of intracellular metabolites under physiological condition.