Despite their inability to reproduce naturally, mules can host embryos and be surrogate dams. The aim of this investigation was to increase our knowledge of the qualitative composition of mule’s milk and its variations throughout the whole lactation period—namely, from 6 h after foaling to 180 days in milk (DIM). Milk was obtained from a mule dam that had foaled after receiving a mule embryo transfer. For each sample, the gross, mineral, and fatty acid composition was evaluated. The average quality of the mule milk was as follows: protein 1.97 g 100 mL⁻¹, fat 0.90 g 100 mL⁻¹, and ash 0.39 g 100 mL⁻¹. Saturated fatty acids made up, on average, 50.00 g 100 g⁻¹ of fat. Monounsaturated and polyunsaturated fatty acids made up half of the total fatty acid content (31.80 g 100 g⁻¹ and 18.2 g 100 g⁻¹ of fat, respectively). Linoleic acid and linolenic acid were the main polyunsaturated fatty acids in the milk. The milk composition changed throughout lactation. Dry matter, protein, fat, and ash decreased significantly from early lactation (6 h to 14 DIM). The n3 polyunsaturated fatty acids decreased at the end of lactation. The changes in milk composition throughout lactation are probably due to adaptations to the growth requirements of the foal.