Photodynamic therapy (PDT) is an effective therapeutic regime for lung cancer. Mitochondrial functional failure is considered to be one of the most important factors causing cell death after PDT. However, the detailed mechanisms that are involved are still unclear. We previously reported that apurinic/apyrimidinic endonuclease (APE1) plays a critical role in regulating sensitivity to PDT in the lung cancer A549 cell line. An important mitochondrial regulatory role for APE1 has recently been reported, so therefore we explored the role of APE1 in cell survival after PDT-induced oxidative stress through regulation of mitochondrial function. We first observed that photoirradiation induced the mitochondrial translocation of APE1. The ability of APE1 to regulate mitochondrial membrane potential and reactive oxygen species (ROS) production after photoirradiation was tested in APE1 knockdown A549 cells. APE1-deficient A549 cells were characterized as having a lower mitochondrial membrane potential and higher ROS production, which led to increased apoptosis through the mitochondrial pathway after PDT. Additionally, unexpected activity of APE1 was observed in mitochondria: the control of mitochondrial transcriptional activity by redox regulation of mitochondrial transcription factor A (TFAM). Furthermore, two dominant-negative mutants of APE1 were overexpressed to enhance their individual activities in mitochondria. The results suggest that both these APE1 activities play a role in the regulation of mitochondrial function but through different mechanisms. The present study not only provides possible mechanisms for APE1 in regulating survival after photoirradiation but also uncovers a new activity of APE1 in mitochondria. (Cancer Sci 2012; 103: 882–888)