We conducted a single-center prospective study to evaluate the utility of cytomegalovirus (CMV) antigenemia assay for the diagnosis of CMV-gastrointestinal disease (GID). The study subjects were HIV-infected patients with CD4 count ≤200 μL/cells who had undergone endoscopy. A definite diagnosis of CMV-GID was made by histological examination of endoscopic biopsied specimen. CMV antigenemia assay (C10/C11 monoclonal antibodies), CD4 count, HIV viral load, history of HAART, and gastrointestinal symptoms as measured by 7-point Likert scale, were assessed on the same day of endoscopy. One hundred cases were selected for analysis, which were derived from 110 cases assessed as at high-risk for CMV-GID after endoscopy screening of 423 patients. Twelve patients were diagnosed with CMV-GID. Among the gastrointestinal symptoms, mean bloody stool score was significantly higher in patients with CMV-GID than in those without (2.5 vs. 1.7, p=0.02). The area under the receiver-operating characteristic curve of antigenemia was 0.80 (95%CI 0.64-0.96). The sensitivity, specificity, positive likelihood ratio (LR), and negative LR of antigenemia were 75.0%, 79.5%, 3.7, and 0.31, respectively, when the cutoff value for antigenemia was ≥1 positive cell per 300,000 granulocytes, and 50%, 92.0%, 5.5, and 0.55, respectively, for ≥5 positive cells per 300,000 granulocytes. In conclusion, CMV antigenemia seems a useful diagnostic test for CMV-GID in patients with HIV infection. The use of ≥5 positive cells per 300,000 granulocytes as a cutoff value was associated with high specificity and high positive LR. Thus, a positive antigenemia assay with positive endoscopic findings should allow the diagnosis of CMV-GID without biopsy.