Objective To explore the relationship between Epstein-Barr virus (EBV) infection with serum human kissin-1 (KISS-1) and matrix metalloproteinase 2 (MMP2) levels and prognosis in patients with colon cancer. Methods A total of 86 colon cancer patients who were hospitalized in our hospital from April 2015 to April 2016 were selected as the colon cancer group; in the same period, 84 cases of physical examination person in our hospital were selected as the control group. Real-time fluorescent quantitative PCR (qRT-PCR) was used to test colon cancer patients for EBV DNA, and divided the patients into EBV DNA negative group and EBV DNA positive group according to the test results. Enzyme-linked immunosorbent (ELISA) method was used to detect serum KISS-1 and MMP2 levels. Pearson method was used to analyze the correlation between serum KISS-1 and MMP2 levels in patients with colon cancer infected with EBV. The survival curve was drawn by Kaplan-Meier method, and the relationship between EBV infection and prognosis of colon cancer patients was analyzed by log-rank test. Multivariate Cox regression analysis were used to analyze the factors affecting the prognosis of colon cancer patients. Results Compared with the control group, the positive rate of EBV DNA in the colon cancer group was higher (χ2=21.854, P<0.001). The EBV DNA positive rate of patients with lymph node metastasis, TNM stage Ⅲ–Ⅳ, tumor low differentiation and tumor infiltration T3–T4 was higher than those without lymph node metastasis, TNM stage Ⅰ–Ⅱ, tumor high/medium differentiation and tumor infiltration T1–T2 (P<0.05). Compared with the EBV DNA negative group, the serum KISS-1 level of the EBV DNA positive group decreased, and the MMP2 level increased (P<0.001). There was a negative correlation between serum KISS-1 and MMP2 levels in colon cancer patients with EBV infection (r=–0.510, P<0.001). The 5-year survival rates of colon cancer patients in the EBV DNA-negative group and the EBV DNA-positive group were 52.94% (27/51) and 14.29% (5/35), respectively, the difference between the two groups was statistically significant (χ2=13.274, P<0.001). EBV infection, MMP2 high expression, and lymph node metastasis were independent risk factors affecting the prognosis of colon cancer patients (P<0.05), and KISS-1 low expression was a protective factor affecting the prognosis of colon cancer patients (P<0.05). Conclusions EBV infection is closely related to the progression and prognosis of colon cancer. The down-regulation of KISS-1 and the up-regulation of MMP2 may be related to EBV infection.