BACKGROUND Serological screening of pregnant women for Toxoplasma gondii-specific antibodies is not practiced as an antenatal care in Tanzania; and there is a limited data about sero-prevalence of T. gondii infection in developing countries. We therefore conducted this study to determine the sero-prevalence and factors associated with T. gondii infection among pregnant women attending antenatal care clinics in Mwanza, Tanzania. METHODS Between 1st November 2012 and 31st May 2013 a total of 350 pregnant women attending antenatal care clinics in Mwanza were enrolled and screened for IgG and IgM antibodies against T. gondii using the ELISA technique. RESULTS Of 350 pregnant women, 108 (30.9%) were sero-positive for T. gondii-specific antibodies. The risk of contracting T. gondii infection increases by 7% with each yearly increase in a woman's age (OR=1.07, 95% CI: 1.02 - 1.11, p=0.002). The sero-positivity rate of T. gondii-specific antibodies was higher among pregnant women from the urban than those from rural communities (41.5% versus 22.0%); [OR=2.2, 95% CI; 1.4 - 3.7, p=0.001]. Likewise employed/business women were more likely to get T. gondii infection than peasants (40.0% versus 25.9%) [OR=1.9, 95% CI: 1.2 - 3.0, p=0.006]. CONCLUSIONS Sero-prevalence of T. gondii-specific antibodies is high among pregnant women in Mwanza with a significant proportion of women at risk of contracting primary T. gondii infections. Screening of T. gondii infections during antenatal care should be considered in Tanzania as the main strategy to minimize congenital toxoplasmosis.