Objective: To understand the prevalence of Toxoplasma gondii infection among special populations in Nanjing City, so as to provide the reference for formulating the interventions for the prevention and control of T. gondii infections in this population., Methods: The HIV/AIDS patients, tumor patients, pregnant women, and people with livestock and poultry breeding or processing works were selected as the study subjects from September to November 2015. The venous blood samples were collected from each participant for detecting IgG and IgM antibodies against T. gondii by ELISA., Results: The overall prevalence of T. gondii infection was 10.2% in the study subjects in Nanjing City. The T. gondii infection rates were 12.2%, 11.3%, 4.0%, and 13.0% among the HIV/AIDS patients, tumor patients, pregnant women, and people with livestock and poultry breeding or processing works, respectively, and there was no statistically significant difference among the four groups ( χ 2 = 5.668, P = 0.130). The prevalence of T. gondii infection was higher in men than in women (15.3% vs. 5.8%; χ 2 = 10.213, P = 0.001), and there were significant differences in the prevalence of T. gondii infection in terms of gender ( χ 2 = 9.501, P = 0.023), education levels ( χ 2 = 9.850, P = 0.043) or occupations ( χ 2 = 8.983, P = 0.062)., Conclusions: The infection rate of T. gondii among the special population in Nanjing City is high. Therefore, the health education intervention should be strengthened in the follow-up work for the special population.