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 tRNA^Leu, thereby facilitating mitochondrial translation. Pathogenic genetic variations in LARS2 are known to cause Perrault syndrome (PS), a disorder characterized by hearing loss and ovarian failure. Here, we determined the crystal structure of LARS2 in complex with leucyl-sulfamoyl-adenylate, and elucidated the molecular basis of its catalytic activity through a structural comparison with its cytosolic counterpart, LARS1. Loss of catalytic activity in LARS2 variants was directly associated with PS pathogenesis, as demonstrated by impaired mitochondrial function. Notably, in a specific LARS2 mutant, overexpression of a tRNA isodecoder partially restored catalytic activity, resulting in an incomplete but significant recovery of mitochondrial function. This study provides crucial insights into the molecular mechanism underlying LARS2-mediated aminoacylation and its essential role in mitochondrial function, as exemplified by PS. Furthermore, our findings suggest that tRNA overexpression could serve as a potential therapeutic strategy for ARS mutation-driven genetic disorders.