DNA fragmentation factor 40 (DFF40), or the caspase-activated DNase (CAD), is an endonuclease specific for double-stranded DNA. Alterations in its function and expression have been linked to apoptosis resistance, a mechanism likely used by cancer cells. Although, how the DFF40-related apoptosis resistance pathway occurs remains unclear. Here we sought to determine if DFF40 could localize to the mitochondrion, a localization not reported in the literature until now, and further regulate cell metabolism when apoptosis is activated. We demonstrated that DFF40 localizes in mitochondria through its N-terminal domain, and its agglomeration is accentuated in apoptosis-activated cells. We also found that a loss of DFF40 expression induces a higher mitochondrial mass, mtDNA copy number, mitochondrial membrane potential, and glycolysis rates in resting T cells. The induction of apoptosis in DFF40 deficient cells doesn’t alter ATP production levels, basal respiration, and mitochondrial membrane integrity. Our study reveals that DFF40 may act as a regulator of mitochondria, and its loss could compromise mitochondrial integrity in pathologies such as cancer.