Purpose Immunocompromised patients may be at risk for reactivation of the toxoplasmosis infection, because of defection in cell-mediated immunity. Therefore, early diagnosis would be highly desirable in these individuals. This case-control study was designed to increase information about toxoplasmosis in hemodialysis (HD) patients in Guilan province, Iran. Methods The study was performed among 150 patients and 150 controls referred to hospitals of Guilan University of Medical Sciences during 2018-2019. Questionnaire forms, including demographic and epidemiological information, were completed. Peripheral blood samples were taken for serum separation and were collected into tubes and then kept at − 20 °C until use. IgG and IgM antibodies to Toxoplasma gondii were detected by a commercial ELISA kit. Accordingly, IgG absorbance levels < 9 were considered negative, 9-11 was considered borderline, and > 11 was positive; IgM absorbance levels < 0.9 were considered negative, 0.9-1.1 was assumed to be borderline, and > 1.1 was positive. Results Throughout the study, 72.0% of HD patients and 64.7% of the control group were positive for anti-Toxoplasma IgG antibody subsequently. 2% of HD patients and 0.7% of the control group were positive for anti-Toxoplasma IgM antibody and these difference weren't significant between control and ones with HD (P > 0.05). There was no significant difference between dialysis duration factor and the seropositivity rate. Seroprevalence of T. gondii infection did not vary significantly with age, educational level, residence and presence of a cat at home. On the contrary, seroprevalence varied significantly with gender and consumption of raw vegetables. Conclusion Because of the high percentage of positivity for Toxoplasma IgG antibodies in hemodialysis patients, we suggest a periodically screening program to carry out for monitoring and evaluating the possible dissemination of toxoplasmosis during hemodialysis.