The repair of DNA damage caused by chemotherapy in cancer cells occurs mainly at two cell cycle checkpoints (G 1 and G 2) and is a factor contributing to chemoresistance. Most colorectal cancers harbor mutations in p53, the main pathway involved in the G 1 checkpoint, and thus, are particularly dependent on the G 2 checkpoint for DNA repair. The present study examined the effect of AZD6738, a specific inhibitor of ataxia telangiectasia mutated and rad3‑related (ATR) involved in the G 2 checkpoint, combined with 5‑fluorouracil (5‑FU), a central chemotherapeutic agent, on colorectal cancer cells. Since 5‑FU has a DNA‑damaging effect, its combination with AZD6738 is likely to enhance the therapeutic effect. The effects of the AZD6738/5‑FU combination were evaluated in various colorectal cancer cells (HT29, SW480, HCT116 and DLD‑1 cells) by flow cytometry (HT29 cells), western blotting (HT29 cells) and water‑soluble tetrazolium 1 assays (HT29, SW480, HCT116 and DLD‑1 cells), as well as in an experimental animal model (HT29 cells). In vitro, the AZD6738/5‑FU combination increased the number of mitotic cells according to flow cytometry, decreased the checkpoint kinase 1 phosphorylation levels and increased cleaved caspase‑3 and phosphorylated form of H2A. X variant histone levels according to western blotting, and decreased the proliferation rate of four colon cancer cell lines according to cell viability experiments. In vivo, xenografted colorectal cancer cells treated with the AZD6738/5‑FU combination exhibited a marked decrease in proliferation compared with the 5‑FU alone group. The present results suggested that AZD6738 enhanced the effect of 5‑FU in p53‑mutated colorectal cancer.