Ribosomes are important cellular components that maintain cellular homeostasis through overall protein synthesis. The nucleolus is a prominent subnuclear structure that contains ribosomal DNA (rDNA) encoding ribosomal RNA (rRNA), an essential component of ribosomes. Despite the significant role of the rDNA‑rRNA‑ribosome axis in cellular homeostasis, the stability of rDNA in the context of the DNA damage response has not been fully investigated. In the present study, the number and morphological changes of nucleolin, a marker of the nucleolus, were examined following ionizing radiation (IR) in order to investigate the impact of DNA damage on nucleolar stability. An increase in the number of nucleoli per cell was found in HCT116 and U2OS cells following IR. Interestingly, the IR‑dependent increase in nucleolar fragmentation was enhanced by p53 deficiency. In addition, the morphological analysis revealed several distinct types of nucleolar fragmentation following IR. The pattern of nucleolar morphology differed between HCT116 and U2OS cells, and the p53 deficiency altered the pattern of nucleolar morphology. Finally, a significant decrease in rRNA synthesis was observed in HCT116 p53‑/‑ cells following IR, suggesting that severe nucleolar fragmentation downregulates rRNA transcription. The findings of the present study suggest that p53 plays a key role in protecting the transcriptional activity of rDNA in response to DNA damage.