The p53-adenovirus vector (Ad-p53) offers a promising approach for cancer therapy, yet its effects on host cell chromatin structure and gene expression are not fully understood. In this study, we employed in situ ChIA-PET to investigate the colorectal cancer cell line HCT116 with p53 knockout, comparing them to cells infected with the adenovirus-vector expressing p53. We examined alterations in chromatin interactions and gene expression following treatment with the anti-cancer drug 5-fluorouracil (5-FU). Our results indicate that Ad-p53 does not impact topologically associating domains (TADs), although gentlely changes in A/B compartments were detected. Ad-p53 mainly interacts with repressive or inactive regions of host chromatin, with no significant effects on overall gene expression. These findings suggest that Ad-p53 boosts p53 expression, enhancing drug sensitivity without substantially altering host HCT116 chromatin architecture.