Plumbagin is a naturally-derived phytochemical which exhibits promising medicinal properties, including anticancer activities. In the present study, the anticancer potential of plumbagin has been demonstrated in lung cancer cells by targeting reactive oxygen species (ROS) and the intrinsic mitochondrial apoptotic pathway. Plumbagin showed impressive cytotoxic, anti-proliferative, and anti-migratory activities with IC50 3.10 ± 0.5 μM and 4.10 ± 0.5 μM in A549 and NCI-H522 cells, respectively. Plumbagin treatment significantly reduced the size of A549 tumor spheroids in a concentration-dependent manner. Plumbagin enhanced ROS production and arrested lung cancer cells in S and G2/M phase. Expression of antioxidant genes such as glutathione S-transferase P1 and superoxide dismutase-2 were found to be upregulated with plumbagin treatment in A549 cells. Plumbagin induced dissipation in mitochondrial membrane potential and affected the expression of intrinsic apoptotic pathway proteins. Increased expression of cytochrome c promotes the activation of pro-apoptotic protein Bax with decreased expression of anti-apoptotic protein Bcl-2. Further, plumbagin activated the mitochondrial downstream pathway protein caspase-9 and caspase-3 leading to apoptosis of A549 cells. Collectively, plumbagin could be a promising future phytotherapeutic candidate for lung cancer treatment via targeting intrinsic mitochondrial apoptotic pathway and ROS.