Cellular senescence refers to an irreversible growth arrest that is triggered by various intrinsic and extrinsic stresses. Many recent studies have demonstrated that cellular senescence plays a crucial role in the regression of tumors exposed to ionizing radiation (IR), but the underlying mechanism remains unknown. Here we show that the activation of integrin β4 is essential for IR-induced cellular senescence. IR treatment results in the phosphorylation of integrin β4 at tyrosine residue 1510, leading to activation of the integrin α6β4-Src-AKT signaling pathway. We further reveal that the IR-induced phosphorylation of integrin β4 is regulated by the cholesterol content and membrane fluidity. We also find that IR-induced p53-caspase signaling is independent of integrin α6β4-Src-AKT signaling. Finally, we show that siRNA- or inhibitor-mediated blockade of integrin α6β4-Src-AKT signaling switches the post-irradiation fate from senescence to apoptosis, under p53 activated condition, in both cancer cells and tumor tissues of xenograft mice. On the basis of our finding that, integrin α6β4 is specifically activated and acts primarily to induce premature senescence in irradiated cancer cells, we propose that this integrin may be a valuable target and biomarker for radiotherapy.