The structure of the human mitochondrial (hs mt) tRNALeu(UUR) features several domains that are predicted to exhibit limited thermodynamic stability. An elevated frequency of disease-related mutations within these domains suggests a link between structural instability and the functional effects of pathogenic mutations. A series of tRNAs featuring mutations within the D and anticodon stems were prepared and investigated using nuclease probing. Structural mapping studies indicated that these domains were partially denatured for the wild type (WT) hs mt tRNALeu(UUR) and were significantly stabilized by mutations introducing additional or stronger base pairs into the stem regions. In addition, trends in the aminoacylation activities of the D stem mutants suggested that the loose structure is required for function, with mutants displaying the most ordered structures exhibiting the lowest levels of aminoacylation activity. A pronounced interdependence of the structures of the anticodon and D stems was observed, with mutations strengthening the D stem stabilizing the anticodon stem and vice versa. The existence of strong interdomain communication was further elucidated with a mutant of hs mt tRNALeu(UUR) containing a stabilized D stem and a pathogenic mutation that disrupted the anticodon stem. Strengthening the structure of the D stem completely restored the function of the disease-related mutant to WT levels, indicating that propagated structural weaknesses contribute to the functional deactivation of this tRNA by mutations.