As a tumor suppressor, p53 preserves genomic integrity in eukaryotes. However, limited evidence is availablefor the p53 shuttling between the cytoplasm and nucleus. Previous studies have shown that β-actin polymerizationnegatively regulates p53 nuclear import through its interaction with p53. In this study, we found thatDNA damage induces both b-actin and p53 accumulation in the nucleus. b-actin knockdown impaired thenuclear transport of p53. Additionally, b-actin could interact with p53 which was enhanced in response togenotoxic stress. Furthermore, N terminal deletion mutants of p53 shows reduced levels of association with β-actin.We further identified Ser15, Thr18 and Ser20 of p53 are critical to the β-actin: p53 interaction, whichupon mutation into alanine abrogates the binding. Taken together, this study reveals that β-actin regulates thenuclear import of p53 through protein–protein interaction.