Colorectal cancer is a common malignancy of the digestive tract with a high mortality rate. However, current treatment approaches such as radiotherapy and chemotherapy are ineffective. Thus, it is imperative to identify ways to treat recurrence and metastasis. The aim of the present study was to investigate the effects of cytokine-induced killer (CIK) cells on the hypoxia microenvironment and immune function in colon 26 cancer xenograft mice. A murine model of colon 26 carcinoma tumors were divided into CIK, normal saline (NS) and control groups. Hypoxia inducible factor-1α (HIF-1α) mRNA in tumor tissue and the small intestine of mice was detected by FQ-PCR. The percentage of the CD4+, CD8+ and CD4+/CD8+ ratio in the spleen of mice was detected by flow cytometry. The tumor volume in the CIK group was smaller than that in the NS group and the difference was not significant (P>0.05). HIF-1α gene expression was present in the tumor tissue and small intestine. HIF-1α gene expression in tumor tissue was significantly higher than that in the small intestine (P<0.05), and lower in tumor tissue of the CIK group compared to the NS group (P<0.05) of xenograft mice. However, no HIF-1α expression was observed in the small intestine of healthy control mice. CD4+ T-cell percentage and CD4+/CD8+ ratio in the spleen of xenograft mice was significantly lower than that of normal mice (P<0.05). Compared to the NS group, the CD4+ T-cell percentage was higher, whereas the CD8+ T-cell percentage was lower in the CIK group (P<0.05). In conclusion, HIF-1α gene expression was present in the tumor tissue and small intestine. The immune function of colon 26 transplanted in tumor mice was decreased. Additionally, CIK improved the microenvironment of tumor hypoxia and promoted immune reconstitution.