Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that catalyze the attachment of amino acids to their cognate tRNAs, enabling protein synthesis. Leucyl-tRNA synthetase 2 (LARS2) is a mitochondrial ARS that specifically ligates leucine to mitochondrial tRNA(Leu), thereby supporting mitochondrial translation. Pathogenic variants in LARS2 cause Perrault syndrome (PRLTS), an autosomal recessive genetic disorder characterized by hearing loss and ovarian failure, for which no pharmacological treatment currently exists. Here, we determined the crystal structure of LARS2 in complex with leucyl-sulfamoyl-adenylate, and elucidated the molecular basis of its catalytic activity through structural comparison with human cytosolic and E. coli leucyl-tRNA synthetases. Disease-associated LARS2 variants exhibited impaired mitochondrial protein synthesis and respiratory function, supporting a pathogenic role in PRLTS. Notably, for the LARS2 R228H variant, exogenous delivery of mitochondrial tRNA(Leu) variant as well as leucine supplementation partially restored catalytic activity, leading to an incomplete but significant recovery of mitochondrial function. This study provides mechanistic insights into LARS2-mediated aminoacylation and its essential role in mitochondrial function. Moreover, our findings highlight the therapeutic potential for substrate-based augmentation as a targeted strategy for treating ARS-related genetic disorders.