To estimate the change in the seroprevalence and risk factors for toxoplasmosis in The Netherlands, a study was conducted in the general population in 2006/2007, similarly designed as a previous study in 1995/1996. Testing 5541 sera for IgG antibodies against Toxoplasma gondii showed a marked decrease of the overall seroprevalence to 26·0% 95% confidence interval (CI) 24·0–28·0, compared to 40·5% (95% CI 37·5–43·4) in 1995/1996. In women of reproductive age the seroprevalence decreased from 35·2% (95% CI 32·9–38·6) in 1995/1996 to 18·5% (95% CI 16·2–20·7) in 2006/2007, leaving the majority of pregnant women susceptible to primary infection with T. gondii and their babies to congenital toxoplasmosis. In participants aged ≥20 years, Toxoplasma seropositivity was associated with living in the Northwest, living in urban areas, low educational level, consumption of raw pork, keeping a cat, and not having occupational contact with clients or patients. For younger participants, risk factors were keeping sheep or cattle, consumption of raw unwashed vegetables and putting sand in the mouth.