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Clinical characteristics of intestinal ulcers complicated with Epstein-Barr virus (EBV) infection remain poorly studied. This study is aimed at providing further insight into clinical features of this patient cohort. The presence of serum EBV DNA was assessed in 399 patients with colonic ulcers, of which 30 cases were positive. In EBV-positive patients, the EBV-encoded RNA (EBER) was detected in intestinal tissues of 13 patients (EBER-positive group). The test was negative in 17 patients (EBER-negative group). Acute EBV infection rate in patients with colonic ulcer was 7.52%. Age and sex differences between two groups were not statistically significant. Fever, abdominal lymph node enlargement, and crater-like gouged ulcer morphology were more common in the EBER-positive group ( P < 0.05 ). The albumin level in the EBER-positive group was significantly lower compared to that in the EBER-negative group ( P < 0.05 ). The copy count of EBV DNA in the blood of patients from the EBER-positive group was higher, and the prognosis was worse ( P < 0.05 ). Clinical manifestations were more severe in the EBER-positive group. Endoscopic, histopathological, and biochemical findings were also more serious in this group of patients. The findings point to the importance of assessing the EBER expression in patients with intestinal ulcers of various etiology. EBER positivity should be viewed as a diagnostic marker of more severe condition requiring more aggressive treatment.