The p53 tumor suppressor plays critical roles in cell cycle regulation and apoptotic cell death, with its activation capable of sensitizing cancer cells to radiotherapy or chemotherapy. To identify small molecules that induce apoptosis via increased p53 transcriptional activity, we used a novel in‐house library containing 96 small‐molecule compounds. Using a cell‐based screening method with a p53‐responsive luciferase‐reporter assay system involving benzoxazole derivatives, we found that AU14022 administration significantly increased p53 transcriptional activity in a concentration‐dependent manner. Treatment with AU14022 increased p53 protein expression, p53 Ser15 phosphorylation, p53‐mediated expression of downstream target genes, and apoptosis in p53‐wild‐type HCT116 human colon cancer cells, but not in p53‐knockout HCT116 cells. Additionally, p53‐wild‐type HCT116 cells treated with AU14022 exhibited mitochondrial dysfunction, including modulated expression of B‐cell lymphoma‐2 family proteins and cytochrome c release. Combination treatment with AU14022 and ionizing radiation (IR) synergistically induced apoptosis as compared with IR or AU14022 treatment alone, with further investigation demonstrating that cell cycle progression was significantly arrested at the G2/M phase following AU14022 treatment. Furthermore, in a mouse p53‐wild‐type HCT116 colon cancer xenograft model, combined treatment with AU14022 and IR inhibited tumor growth more effectively than radiation alone. Therefore, AU14022 treatment induced apoptosis through p53‐mediated cell cycle arrest involving mitochondrial dysfunction, leading to enhanced radiosensitivity in colon cancer cells. These results provide a basis for further assessments of AU14022 as a promising anticancer agent. This article is protected by copyright. All rights reserved