We assessed the seroprevalence of Entamoeba histolytica infection using indirect hemagglutination antibody (IHA) assay among 667 HIV-infected persons (group A), 1,311 asymptomatic HIV-uninfected persons seeking anonymous HIV testing (group B), 616 HIV-uninfected controls with gastrointestinal symptoms (diarrhea and/or liver abscess) seeking medical care (group C), and 2,500 healthy controls undergoing health check-up (group D). An IHA titer >or= 128 was detected in 7.1% of group A, 2.5% of group B, 1.8% of group C, and 0.1% of group D (P < 0.0001). The highest seroprevalence (11.2%) was noted among HIV-infected persons who were men having sex with men (MSM) 30-39 years of age. Compared with persons with gastrointestinal symptoms, the adjusted odds ratio for having high IHA titers among HIV-infected persons was 3.206 (95% confidence interval, 1.433, 7.176; P = 0.005). These findings show that HIV-infected persons, especially MSM 30-39 years of age, are at significantly higher risk of E. histolytica infection.