The protozoon Toxoplasma gondii has a worldwide distribution and affects many species of warm-blooded animals. In the Canadian prairies, mesocarnivores such as striped skunks (Mephitis mephitis) and raccoons (Procyon lotor) have experienced an increase in density and distribution, and they are in close contact with human dwellings. However, there has been no systematic study on the seroprevalence of T. gondii in these mesocarnivore populations. The objectives of the current project were to determine the serum antibody prevalence of T. gondii in Canadian prairie mesocarnivores and to study the relationship between antibody prevalence and species, sex, age, location, and year of collection. Antibodies to T. gondii were found in 5 of 24 (20.8%) skunks from Saskatchewan trapped in 1999 and 5 of 40 (12.5%) in 2000. Seroprevalences for T. gondii in raccoons and skunks trapped in Manitoba were 2 of 10 (20%) raccoons trapped in 2002, 7 of 44 (15.9%) trapped in 2003, and 16 of 37 (43.2%) trapped in 2004; and in 13 of 99 (13.1%) skunks trapped in 2003, 29 of 131 (22.1%) trapped in 2004, 53 of 165 (32.1%) trapped in 2005, and 30 of 51 (58.8%) trapped in 2006. Age, location, and year, but not the host species, were important variables in the determining the seroprevalence of T. gondii in skunks and raccoons. Results confirm that T. gondii is endemic in the skunk and raccoon populations in the Canadian prairies.