Abstract Background Toxoplasmosis during pregnancy can result in congenital malformations and fetal death. In France, universal antenatal screening and monthly re-testing of seronegative pregnant women has been established since the 1970s. However, decreasing Toxoplasma gondii seroprevalence among pregnant women and lower incidence of congenital toxoplasmosis may challenge the cost-effectiveness of such programs. The aim of this study was to estimate the current seroprevalence among pregnant women participating in the 2021 national perinatal survey (ENP). Methods All adult women giving birth in France between 15-21 March 2021 were invited to participate in the ENP. Data collected included demographic information, nationality, socio-economic status, education level and Toxoplasma gondii serology. We classified a woman as seropositive if she had IgG antibodies prior to pregnancy or seroconverted during it. Univariable and multivariable regression analyses using Poisson model were conducted to estimate prevalence ratios (PRs) and identify significant factors associated with seropositivity. Results Among the 15,605 participating women, 0.22% seroconverted during pregnancy. The overall seroprevalence was 25.95%. Seropositivity was higher with increasing age (∼5% per 5 years), among residents of French overseas territories Guadeloupe/Saint Martin (PRs:1.07; 95%-CI: 1.03-1.10), La Réunion (PRs:1.10; 95%-CI: 1.08-1.13) and Mayotte (PRs:1.27; 95%-CI: 1.23-1.31), with lower education (PRs:1.22; 95%-CI: 1.04-1.42) and African nationality (PRs:1.11; 95%-CI: 1.02-1.20). Conclusions High seroprevalence for Toxoplasma gondii was found in older women, which may reflect their higher risk of past exposure. The observed geographical differences in seroprevalence may mirror dietary and environmental diversity. Declining seroprevalence among pregnant women in France can affect screening effectiveness and warrants a comprehensive review to determine appropriate future prevention strategies. Key messages • Older women and residents of French overseas territories show higher toxoplasmosis seroprevalence, possibly due to past exposure risks and diverse diets/environments. • Declining toxoplasmosis seroprevalence among pregnant women in France can affect screening effectiveness and warrants a comprehensive review to determine appropriate future prevention strategies.