Targeting cell cycle has become one of the major challenges in cancer therapy, being Palbociclib, a CDK4/6 inhibitor, an excellent example. Recently, it has been reported that Palbociclib could be a novel radiosensitizer agent. In an attempt to clarify the molecular basis of this effect we have used cell lines from colorectal (HT29, HCT116) lung (A549, H1299) and breast cancer (MCF-7). Our results indicate that the presence of a p53 wild type is strictly required for Palbociclib to exert its radiosensitizing effect, independently of the inhibitory effect exerted on CDK4/6. In fact, abrogation of p53 in cells with functional p53 blocks the radiosensitizing effect of Palbociclib. Moreover, no radiosensitizing effect is observed in cells with non-functional p53, but restoration of p53 function promotes radiosensitivity associated to Palbociclib. Furthermore, the presence of Palbociclib blocks the transcriptional activity of p53 in an ATM-dependent-fashion after ionizing radiation exposure, as the blockage of p21/WAF1 expression demonstrates. These observations are a proof of concept for a more selective therapy, based on the combination of CDK4/6 inhibition and radiotherapy, which would only benefit to those patients with a functional p53 pathway.