<p>Adenine nucleotide translocase 1 (ANT1), involved in exchanging ATP and ADP across the mitochondrial inner membrane, is downregulated in mouse brains with Parkinsonian variations. To further explore the role of ANT1 in neuronal cells, an intensive investigation was conducted by introducing overexpressed ANT1 and ANT1 mutant at Asn177 into neuroblastoma SH-SY5Y cells treated with MPP⁺. Consequently, ANT1 was found to be involved in maintaining mitochondrial functions by attenuating ROS levels and ameliorating a long-lasting mPTPs opening and aberrant mitochondrial membrane potential (△Ψm) induced by MPP⁺. RNA-Seq analysis revealed that the processes including respiration, mitochondrial transporting, mitochondrial organization and apoptosis were highly facilitated in response to ANT1 supplement under MPP⁺ treatment. Additionally, ANT1 enrichment promoted a clearance of the damaged cells via activating the DDIT3-CytC-related pathway and resulted in an intensified structure of actin microfilaments. However, ANT1 mutant served as a causative factor, since it led to mitochondrial dysfunction via promoting a long-lasting mPTPs opening, inactivating DDIT3-CytC-related pathway and strongly impairing actin microfilaments. These observations are helpful to improve the understanding of the role of ANT1 in regulating mitochondrial functions in neuronal cells and to explore a potential therapeutic implication of ANT1 for Parkinson’s disease as a promising target.</p>