Toxoplasma gondii (T. gondii), an obligate intracellular protozoan parasite is a known etiological factor of reproductive problems and encephalomyelitis in animals and humans. This study investigated the seroprevalence of T gondii infection in recreational horses in two metropolitan cities of southwestern Nigeria. Serum samples were randomly collected from a total of 157 horses in Lagos and Ibadan. Samples were assayed for the presence of T gondii antibodies using the Modified Agglutination Test (MAT). A total of 22 (14%) of the 157 sampled horses were positive for T gondii antibodies by MAT with titers of 1:20 in 12 samples (54.5%), 1:40 in 8 samples (36.4%), 1:80 in 1 sample (4.1%) and 1:160 in 1 sample (4.1%). Seroprevalence varied among gender, breeds, age groups and sampling locations but there was no statistically significant association (p < 0.05) of any of the factors to T. gondii infection. This study showed that recreational horses in southwestern Nigeria are exposed to T. gondii and appropriate measures should be adopted to prevent and control the infection in horses in this region. The zoonotic implication of the disease is also discussed. This is probably the first report on seroprevalence of Toxoplasma gondii in horses in southwestern Nigeria.