Dysregulation of the immune system may play important roles in the development of colorectal cancer (CRC). The C-X-C chemokine receptor type 5 (CXCR5) is one of the principal regulators for targeting T cells, B cells, and dendritic cells into secondary lymphoid organs. The current study investigated the association between CXCR5 gene polymorphisms and the risk of CRC, and the potential effect of these polymorphisms on different immune cells. Two polymorphisms in CXCR5 gene, rs6421571C/T and rs80202369G/A, were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 302 cases and 316 controls. Results showed that individuals with the rs6421571CT and TT genotypes had a strong correlation with the incidence of CRC (odds ratio (OR) = 1.46; 95 % confidence interval (CI), 1.02-2.09; p = 0.041 and OR = 2.62; 95 % CI, 1.50-4.95; p < 0.001, respectively). Also, rs80202369AA genotype revealed significantly higher distribution in CRC patients than in controls (p = 0.002). We further investigated the possible effects of the polymorphisms by assessing messenger RNA (mRNA) and protein levels of CXCR5 in peripheral blood mononuclear cells (PBMCs), CD4+ T cells, CD8+ T cells, and B cells. Data presented that healthy controls with rs6421571CT and TT genotypes had higher mRNA and protein levels of CXCR5 than those with wild-type CC genotype specifically in CD4+ T cells. These findings suggest novel risk factors of CRC and indicate a potential mechanism of CXCR5 gene polymorphism.