The current study examines 2-deoxy-D-glucose (2-DG), a non-metabolizable glucose analog, for its potential apoptotic effect via reactive oxygen species (ROS) generation and inhibition of antioxidant system in A549 cell line (lung cancer). The cytotoxicity, annexin-V/propidium iodide (PI), ROS at intracellular levels, caspase-3 activity, mitochondrial membrane potential (MMP), ATP level, DNA fragmentation and antioxidants were examined in A549 cells exposed to 2-DG (2–10 mM) for 24 h. The cytotoxicity effect was also measured in the normal human embryonic kidney (HEK293) cell line. The outcomes indicated a concentration-dependent cytotoxic effect of 2-DG and favored apoptotic cell death of the A549 cells with non-significant effect in HEK293 cells. 2-DG caused morphological and cellular density changes, with annexin V/PI uptake and MMP reduction in the A549 cells confirming apoptotic cell death. Additionally, depletion of ATP at intracellular level, elevated DNA fragmentation and caspase-3 activity were observed, indicating caspase-3 dependent apoptosis. To understand if 2-DG mediates apoptosis via ROS production, the A549 cells was pre-treated with α-tocopherol, an ROS inhibitor, followed by 2-DG treatment. Interestingly, the cytotoxicity and generation of ROS in α-tocopherol-pretreated A549 cells was lowered significantly when compared to the untreated A549 cells indicating that 2-DG stimulated apoptosis is mediated via ROS production. Several anticancer medications are characterized by their capacity to increase ROS which are controversial as they damage normal cells. However, cancer cells are susceptible to using 2-DG more than the healthy normal cells making it a suitable anti-cancer drug with a scope to be used against resistant lung cancer.