A 25-d trial was performed to assess the effects of machine and hand milking on behavior, and milk yield and composition of mares; the trial involved 8 Murgese mares. Milk yield was higher (7.69 vs. 4.91 kg) and milking time was shorter (1.80 vs. 5.40 min) in machine-milked than in hand-milked mares. Machine milking of mares also resulted in a greater fat content of milk (1.63%) than did hand milking (1.06%). Milking system did not affect casein content, nitrogen fractions, or somatic cell counts. No differences in behavioral activities and in response to humans emerged. Results indicate that machine milking of mares could improve the yield and gross composition of horse milk, without adverse consequences on mare behavior and response to humans.