Six pathogenic mutations have been reported in human mitochondrial tRNA(Thr) (hmtRNA(Thr)); however, the pathogenic molecular mechanism remains unclear. Previously, we established an activity assay system for human mitochondrial threonyl-tRNA synthetase (hmThrRS). In the present study, we surveyed the structural and enzymatic effects of pathogenic mutations in hmtRNA(Thr) and then focused on m.15915 G > A (G30A) and m.15923A > G (A38G). The harmful evolutionary gain of non-Watson-Crick base pair A29/C41 caused hmtRNA(Thr) to be highly susceptible to mutations disrupting the G30-C40 base pair in various ways; for example, structural integrity maintenance, modification and aminoacylation of tRNA(Thr), and editing mischarged tRNA(Thr). A similar phenomenon was observed for hmtRNA(Trp) with an A29/C41 non-Watson-Crick base pair, but not in bovine mtRNA(Thr) with a natural G29-C41 base pair. The A38G mutation caused a severe reduction in Thr-acceptance and editing of hmThrRS. Importantly, A38 is a nucleotide determinant for the t6A modification at A37, which is essential for the coding properties of hmtRNA(Thr). In summary, our results revealed the crucial role of the G30-C40 base pair in maintaining the proper structure and function of hmtRNA(Thr) because of A29/C41 non-Watson-Crick base pair and explained the molecular outcome of pathogenic G30A and A38G mutations.