AbstractAutism spectrum disorder (ASD) is a neurodevelopmental condition often associated with mitochondrial dysfunction, including increased mitochondrial DNA (mtDNA) copy number and impaired energy production. This study investigates the role of the mitochondrial replisome—specifically, the genes TFAM, TWNK, POLG, and TOP1MT—in mtDNA replication and its potential contribution to ASD pathophysiology. We analyzed samples from the oral mucosa of children with ASD and typically developing (TD) controls, assessing mtDNA copy number, gene expression, and protein levels. Our findings revealed a significant increase in mtDNA copy number in the oral mucosa of ASD children, along with partially deleted mtDNA molecules. However, there were no significant changes in the expression of TFAM, TWNK, POLG, or MT‐TL1 genes between ASD and TD samples. Additionally, TFAM protein levels, including monomeric, dimeric, and trimeric forms, did not differ significantly. We also observed increased oxidative stress and inflammatory markers in the oral mucosa of ASD children, suggesting that mitochondrial alterations may be linked to inflammation and oxidative damage in ASD. To further investigate the functional impact of TFAM, we overexpressed it in human HEK293 cells and cortical neurons (CN1.4). TFAM overexpression led to increased mtDNA copy number, cell proliferation, and ATP production in HEK293 cells, but did not significantly alter mitochondrial gene expression, protein oxidation, or mtDNA integrity. In CN1.4 neurons, TFAM overexpression increased mitochondrial membrane potential and length, indicating potential changes in mitochondrial dynamics. Overall, our study suggests that while mtDNA alterations are present in ASD, they are not directly driven by changes in mitochondrial replisome gene expression. These findings highlight the complexity of mitochondrial dysfunction in ASD and suggest the need for further investigation into the underlying molecular mechanisms.