Certain mutations in mitochondrial DNA (mtDNA) are associated with Leber's hereditary optic neuropathy (LHON). In particular, the well‑known NADH dehydrogenase 4 (ND4) m.11778G>A mutation is one of the most common LHON‑associated primary mutations worldwide. However, how specific mtDNA mutations, or variants, affect LHON penetrance is not fully understood. The aim of the current study was to explore the relationship between mtDNA mutations and LHON, and to provide useful information for early detection and prevention of this disease. Following the molecular characterization of a Han Chinese family with maternally inherited LHON, four out of eight matrilineal relatives demonstrated varying degrees of both visual impairment and age of onset. Through PCR amplification of mitochondrial genomes and direct Sanger sequencing analysis, a homoplasmic mitochondrial‑encoded ND4 m.11778G>A mutation, alongside a set of genetic variations belonging to human mtDNA haplogroup B5b1 were identified. Among these sequence variants, alanine transfer RNA (tRNA)Ala m.5601C>T was of particular interest. This variant occurred at position 59 in the TψC loop and altered the base pairing, which led to mitochondrial RNA (mt‑RNA) metabolism failure and defects in mitochondrial protein synthesis. Bioinformatics analysis suggested that the m.5601C>T variant altered tRNAAla structure. Therefore, impaired mitochondrial functions caused by the ND4 m.11778G>A mutation may be enhanced by the mt‑tRNAAla m.5601C>T variant. These findings suggested that the tRNAAla m.5601C>T variant might modulate the clinical manifestation of the LHON‑associated primary mutation.