Background: F1Fo-ATP synthase (F1Fo-ATPase) is a reversibly rotary molecular machine whose dual functions of synthesizing or hydrolyzing ATP switch upon the condition of cell physiology. The robust ATP-hydrolyzing activity occurs in ischemia for maintaining the transmembrane proton motive force of mitochondria inner membrane, but the effect of F1Fo-ATPase on X-ray response of non-small-cell lung cancer (NSCLC) cells is unknown. Methods and Findings: We studied whether ATP hydrolysis affected X-ray radiation induced cell death. NSCLC cells (A549) were pretreated with BTB06584 (BTB), an elective ATP hydrolysis inhibitor, followed by X-ray radiation. Cell viability and clonogenic survival were markedly decreased, clear indications of enhanced radiosensitivity through BTB incubation. Additionally, ATP5a1 was upregulated in parallel with elevated ATP hydrolytic activity after X-ray radiation, showing an increased mitochondrial membrane potential (Delta psi(m)). ATP hydrolysis inhibition led to collapse of Delta psi(m) suggesting ATP hydrolytic activity could enhance Delta psi(m) after X-ray radiation. Furthermore, we also demonstrated that apoptosis was pronounced with the prolonged collapse of Delta psi(m) due to hydrolysis inhibition by BTB incubation. Conclusion: Overall, these findings supported that ATP hydrolysis inhibition could enhance the radiosensitivity in NSCLC cells (A549) after X-ray radiation, which was due to the collapse of Delta psi(m).