This case-control study aimed to characterize the factors associated with amebiasis, defined as presence of anti-Entamoeba histolytica antibody titers of ≧ 128 by indirect hemagglutination assay, among persons seeking voluntary counseling and testing (VCT) for human immunodeficiency virus (HIV) infection. Between April 2006 and September 2009, 57 of 4,802 persons (1.2%) seeking VCT services were seropositive for E. histolytica infection. Compared with 228 seronegative controls, case subjects were older (odds ratio [OR] for per 1-year increase, 1.098; 95% confidence interval [CI], 1.036, 1.165), less likely to hold bachelor degree or higher (OR, 0.359; 95% CI, 0.152, 0.846), and were more likely to be men who have sex with men (MSM) (OR, 8.382; 95% CI, 2.050, 34.266) and have oral-anal sex (OR, 4.016; 95% CI, 1.711, 9.427) in multiple logistic regression analysis. The MSM, fecal-oral contamination, lower educational achievement, and older age were associated with increased risk for amebiasis among persons seeking VCT for HIV infection.