MTO1, together with MSS1 and MTO2, is a gene involved in the pathway of encoding a mitochondria-specific RNA-modifying enzyme related to the post-transcriptional modification of mitochondrial tRNAs. We have previously shown that a mutation of the MTO2 or MSS1 gene can suppress the neomycin-sensitive phenotype of yeast carrying a mitochondrial 15S rRNA C1477G mutation. Here we report that a null mutation of MTO1 also can inhibit the aminoglycoside-sensitivity of yeast carrying mitochondrial 15S rRNA C1477G mutation. The C1477G mutation corresponds to the human 12S rRNA A1555G mutation. Yeast with an mtDNA C1477G mutation exhibits hypersensitivity to neomycin and displays mitochondrial function impairment beyond neomycin treatment. When the mto1 null mutation and mitochondrial C1477G mutation coexist, the yeast strain shows growth recovery. The deletion of the nuclear gene MTO1 regulates neomycin sensitivity in yeast carrying the mitochondrial 15S rRNA C1477G mutation. MTO1 deletion causes the expression levels of the key glycolytic genes HXK2, PFK1 and PYK1 to become significantly up-regulated. The energy deficit due to impaired mitochondrial function was partially compensated by the energy generated by glycolysis. Being in the same pathway, the regulation of MTO1, MSS1 and MTO2 to the neomycin-sensitivity of yeast showed difference in the growth activity of strains, mitochondrial function and the expression level of glycolytic genes.