The presence of antibodies to Toxoplasma gondii in livestock and poultry was investigated by latex agglutination tests; samples that agglutinated at dilutions of 1:64 or higher were regarded as positive. Sera were collected from fattening beef cattle (102 Japanese black, 105 crossbreeds and 114 castrated Holstein), culled dairy cattle (101 Holstein), 100 horses, 115 fattening pigs and 235 chickens (163 free-range and 72 broilers) at abattoirs in Gifu Prefecture, Japan, from August 2012 to August 2013. Antibodies to T. gondii were found in 7.3% (31/422) in cattle, 5.2% (8/155) in pigs, but not in horses or chickens. These results suggest that toxoplasmosis may be transmitted to humans via consumption of T. gondii-infected raw beef in Japan.