Background: Toxoplasmosis is a worldwide disease caused by an obligate intracellular protozoan, Toxoplasma gondii and can cause severe infections in immune-compromised individuals. Objectives: The aim of this study was to determinate the anti-Toxoplasma antibodies in hemodialysis patients of Abadan and Khoramshahr, Southwest of Iran. Materials and Methods: Sera of 150 patients (test group) aged 21 to 87 years referred regularly to hemodialysis departments in Abadan and Khoramshahr cities, and 150 healthy individuals (control group) were examined for anti-Toxoplasma (IgG and IgM) antibodies using ELISA kits and the results were analyzed using Chi-square and fisher exact test. Results: 61 (40.67%) out of 150 sera of patients were positive, 6 (4%) were borderline and 83 (55.33%) were negative for anti-Toxoplasma IgG. For anti-Toxoplasma IgM, 13 (8.67%) of 150 were positive, 21 (14%) were borderline and 116 (77.33%) were negative. In control group 39 (26%) of 150 individuals were positive, 14 (9.33%) were borderline and 97 (64.67%) were negative for anti-Toxoplasma IgG. In the sera of individuals in control group, anti-Toxoplasma IgM was not detected. In hemodialysis patients, 7 (4.66%) cases were positive for anti-Toxoplasma IgG and IgM and 8 (5.33%) cases were IgG positive and IgM borderline. There were significant differences in IgG and IgM level of the test and control groups (P ˂ 0.05) Conclusions: Hemodialysis patients are high risk group for toxoplasmosis and should be tested periodically to prevent the dissemination of toxoplasmosis during dialysis.