Simple SummaryDonkey milk, because of its nutritional quality, is a more interesting product for the human diet. Moreover, due to its similarity to human milk, it is an optimal substitute for breast milk for babies. The aim of this study is to provide new insights on donkey milk production, which characterizes gross and mineral composition of Ragusano donkey breed milk. Compared to cow milk, donkey milk is characterized by a lower content of dry matter, high lactose amount, low protein, and a very low-fat content. In addition, the casein content is lower than cow’s milk. The most abundant macro element was K, followed by Ca, Na, and P. Most of the constituents are diminishing their content during lactation. In conclusion, the Ragusano breed showed a good aptitude for milk production. The composition of the milk confirmed its nutritional quality. Moreover, this research can have important positive impacts. The Ragusano breed has a long lactation and a high level of production that can ensure good income for farmers. Moreover, given its nutritional values, a larger consumption of donkey milk would be desirable and could promote the breeding of the donkey, which is an endangered species all over the world, as a profitable alternative for farmers.Donkey milk is increasingly being proposed as a natural alternative milk for various categories of consumers, especially infants and the elderly population. However, its potential production, gross, and mineral composition have not been deeply investigated yet. Sixty-two individual milk samples were collected monthly from nine Ragusano donkeys reared in a specialized dairy farm. Milk yield as well as chemical and mineral composition, including macro and micro elements, were investigated over an entire lactation, from the second to the ninth month of milking. Milk yield averaged 1.64 kg/day, which highlights good aptitude of the Ragusano breed for the production of milk. Gross composition was characterized by low content of dry matter (8.19%), a high amount of lactose (6.07%), low protein (1.34%), and very low-fat content (0.16%). Whey proteins represented 58% of the total protein, and proteose-peptones accounted for 0.35 ± 0.07 g per 100 g. Total ash content was 0.36 g per 100 g and represented 4.40% of the dry matter. The most abundant element was K, which was followed by Ca, Na, and P. As expected, the micro elements Fe, Zn, and Cu were found in low amounts or in traces. Dry matter, fat, whey proteins. The total ash, Ca, P, Mg, and mineral ratios were significantly affected by the lactation stage.