Toxoplasmosis is a zoonosis caused by infection with Toxoplasma gondii and is prevalent worldwide under various climatic conditions. It is usually asymptomatic, but infection in pregnant women can pose serious health problems for the fetus. However, epidemiological information regarding toxoplasmosis in Japanese pregnant women is limited. This study aimed to determine the prevalence of anti-Toxoplasma antibodies, the primary infection rate, and the risk factors for toxoplasmosis in Japanese pregnant women. We measured anti-Toxoplasma antibody titers in 4,466 pregnant women over a period of 7.5 years and simultaneously conducted interviews to identify the risk factors for toxoplasmosis. The overall prevalence of anti-Toxoplasma antibodies was 10.3%, and it was significantly higher in women aged above 35 years. The rate of primary Toxoplasma infection during pregnancy was estimated to be 0.25%. A possibility of infection in the later stages of pregnancy was identified for those women who were not infected in the early stages. A history of raw meat intake was identified to be a risk factor related to toxoplasmosis. Therefore, to lower the risk of toxoplasmosis, pregnant women should refrain from eating raw and undercooked meat and maintain personal hygiene.