The decrease in the copy number of mitochondrial DNA (mtDNA) in cancer tissues might be associated with a decrease in oxidative mtDNA damage to achieve cancer immortalization and progression. Lung cancer specimens were collected from 29 patients with stage III non-small cell lung cancer (NSCLC) after neoadjuvant chemotherapy followed by surgical resection. The relative mtDNA copy number and the oxidative mtDNA damage (formation of 8-OHdG in mtDNA) of each cancer tissue were measured by quantitative real-time PCR. Seven female and 22 male lung cancer patients, with a mean age of 63.5 years were evaluated. Tumors of five patients became progressive, 13 stable, and 11 partially responsive after preoperative chemotherapy. Low mtDNA copy number (P=0.089) and low degree of oxidative mtDNA damage (P=0.036) were found to associate with tumor progression. Moreover, mtDNA copy number was significantly related to the degree of oxidative mtDNA damage (P=0.031). The mtDNA copy number and oxidative mtDNA damage were lower in advanced NSCLC after chemotherapy. This finding suggests that a decrease in the content of mtDNA may result in a decrease of mitochondrial density in cancer cells, which leads to a decrease of endogenous ROS production and reduction of ROS-triggered DNA damage to achieve immortalization.