Fruit ripening is characterized by dramatic changes in fruit quality and is considered to influence postharvest storage. The mitochondria are one of the most important organelles that play crucial roles in fruit quality formation, but the underlying mechanisms remain unclear. In this study, the expression profiles of mitochondrial proteins during tomato fruit ripening were investigated using quantitative proteomic analysis, leading to the identification of 27 proteins which showed altered abundance. We additionally evaluated changes in the mitochondrial proteome in the ripening-deficient mutant, ripening-inhibitor (rin), which carries a mutation in the transcription factor RIN. A total of 74 proteins were identified which changed abundance significantly in the rin mutant. Gene expression analysis combined with chromatin immunoprecipitation indicated that four genes, DnaK, HSP, PYP, and ACC, which encode chaperone DnaK, heat shock protein, pyruvate kinase, and acetyl-CoA carboxylase, respectively, are direct targets of RIN. These results suggest that mitochondria might participate in the regulation of fruit quality and ripening by specific proteins. Our findings provide new information to unravel of gene regulatory networks that control fruit quality and ripening.