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Toxoplasma gondii has been found to infect almost all warm-blooded animals, including humans. In this study, a total of 3,275 human serum samples were collected from hospitals in five provinces of China. About 5.13% (168/3,275) (95% CI, 4.42–5.94) of the serum samples tested positive for T. gondii IgG antibody by a modified agglutination test (MAT) (cut-off: 1:20). Significant associations were detected between geographic location (OR = 1.763), age (OR = 3.072), infertility in women (OR = 2.4409) and T. gondii infection in humans (p < 0.05). To minimize infection, citizens need to be informed about the best practices for toxoplasmosis prevention, including eating well-cooked meat, drinking boiled water, washing vegetables and fruits, and being careful during contact with cats.