Previously, we have shown that a combination of metabolic modifiers 2-deoxy-d-glucose (2-DG) and 6-aminonicotinamide (6-AN) results in oxidative stress mediated radiosensitization of malignant cells via mitochondrial dysfunction and non-coordinated expression of antioxidant defense, besides inhibition of repair and recovery. In the present study, our objective was to study, in a panel of human malignant cells of various origins (lung carcinoma, squamous carcinoma, oral carcinoma, and glioblastoma), if the inhibitory activity of combination (2-DG+6-AN+2Gy) against tumor growth could be considered a general phenomenon and to determine its effect on the cell cycle. The results revealed that combination (2-DG+6-AN+2Gy) treatment result in significant cell growth inhibition and induced ROS generation in all cancer cells studied. The anti-proliferative effect was related to the ability of combination (2-DG+6-AN+2Gy) to provoke growth inhibition at the G2/M arrest and apoptosis. Furthermore, combination (2-DG+6-AN+2Gy) induced G2/M arrest is closely correlated to decreased cyclin A, cyclin B1, and cdc2 levels.