Toxoplasmosis, an important zoonosis, can be transmitted by eating meat or drinking milk of animals infected with Toxoplasma gondii. Samples were collected from 238 donkeys in the year 2010 in Italy, which included 207 females and 31 males of five breeds and crossbreeds with the average age 9 years (1 month-24 years). Sera were tested for T. gondii antibodies using a latex agglutination test and the indirect fluorescent antibody test; 5 and 8% seropositivity were recorded, respectively. We found significant correlation between the presence of T. gondii antibodies and sex, age, grazing and presence of cats on the farms and their access to donkey feed. This is the first detection of T. gondii antibodies in donkeys in Italy.