The seroprevalence of Toxoplasma gondii infection in horses in Mexico is unknown. Therefore, antibodies to T. gondii were assessed in 495 horses in Durango State, Mexico, using the modified agglutination test (MAT). Horses were from 18 farms in 3 municipalities in the valley region of Durango State. Antibodies to T. gondii were found in 30 (6.1%) of 495 horses, with MAT titers of 1∶25 in 19, 1∶50 in 6, and 1∶100 in 5. Seropositive horses were found in 8 (44.4%) of 18 farms sampled in all 3 municipalities. Age, gender, and breed of horses did not influence significantly the seroprevalence of T. gondii infection. The seroprevalence in horses was higher in rural (7.8%) versus urban (0%) areas, and in larger herds (30–64 horses, 7.8%) versus smaller ones (3–28, 3.5%). The seroprevalence was higher in stall fed (16.5%) than in pastured horses (3.1%). Results indicate that infected horses represent a potential source of T. gondii infection for humans in Mexico. This is the first report of T. gondii infection in horses in Mexico.