Earlier, we reported that Demethoxycurcumin (DMC) suppressed the growth of human glioma U87 MG cells by downregulation of Bcl-2 expression. In the present work, we investigated the DMC induced reactive oxygen species (ROS) mediated anti-proliferative and apoptotic effects in U87 MG cells. Exposure of U87 MG cells to growth-suppressive concentrations of DMC (0–50μg/ml) resulted in ROS generation and concomitant increase in apoptosis. The major oxidative species induced by DMC was superoxide anion radical (O2−•). DMC-induced anti-proliferation was mediated by Akt/NF-κB signalling inhibition and apoptosis through caspase-8 and 9 activation. In silico molecular docking analysis showed that, the amino acid residues His30, Tyr34, Asn37, Ala63, Asn67, His74, Trp123, and Asp159 in the active site of mitochondrial SOD (MnSOD) interacted with DMC. Furthermore, the complex MnSOD-DMC was found to be more stable as compared to native MnSOD in the MD simulations. In the present study, we have demonstrated for the first time using U87 MG cell line that DMC (a) establishes π-π interactions with Tyr 34 and Trp 161 in the putative active site of MnSOD to inhibit its activity, generating (O2−•) to regulate survival and apoptotic proteins leading to antiproliferative and apoptotic events (b) induces antiproliferative effect via inhibition of Akt/NF-κB signalling pathway (c) contributes to the apoptosis via caspase-8 and caspase-9 activation to release the cytochrome c. In exploring the DMC induced cell death events in U 87 MG cell line, we revealed a novel mechanism of DMC-mediated inhibition of MnSOD leading to accumulation of superoxide anions to trigger the inhibition of survival pathways and induction of apoptosis.