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Vitamin D deficiency is associated with high susceptibility to infections. The present study aimed at exploring the relationship between vitamin D levels and Toxoplasma gondii ( T. gondii ) infection, on the basis of a nationally representative database. The study data came from the National Health and Nutrition Examination Surveys (NHANES) 2001–2004. Participants underwent both Toxoplasma IgG antibody testing and serum vitamin D testing. Vitamin D deficiency was defined by a serum 25-hydroxyvitamin D level <20 ng/mL. Multivariate logistic regression and propensity score matching were used to adjust for potential confounders. All analyses were conducted in R software. A total of 10613 participants were included. Among these, 3973 (37.4%) were vitamin D deficient, and 2070 (19.5%) were seropositive for T. gondii IgG antibody. Vitamin D deficiency was found in 42.3% of the seropositive population, compared with 36.3% of the seronegative population ( P <0.001). After adjustment for sex, age, body mass index, smoking history, drinking history and testing season, vitamin D deficiency was associated with an elevated risk of T. gondii infection (OR=1.303, 95% CI=1.136–1.495, P <0.001). This effect persisted in the propensity matching cohort. Low vitamin D levels are associated with high seroprevalence of T. gondii .