Simple SummaryDonkey milk, a functional food, can be used as a milk replacement for newborn nutrition, due to its similar chemical composition to maternal breast milk and hypoallergenic property, and may be useful in the prevention of hypercholesterolemia and atherosclerosis. However, donkey milk yield is very low and cannot satisfy the demands of the market. Some research on dairy cows showed that increasing dietary crude protein levels can increase milk yield and milk component yields. Therefore, this study explored whether increasing dietary crude protein levels could promote the milk production of lactating donkeys. The results showed that increasing crude protein levels could improve milk performance and nutrient digestibility of lactating donkeys. The key finding of this study was that a diet containing 14.2% crude protein supplied adequate protein to improve milk production in lactating donkeys.Donkey milk is considered as a functional food due to its high levels of whey protein, and can be used in newborn nutrition, due to the nutritional similarities with human milk and its hypoallergenic property. However, donkey milk yield is very low and little is known about improving donkey milk yield by nutrition manipulation. The effect of dietary crude protein (CP) levels on milk production, nutrient digestibility, and serum metabolites was investigated in the current study. Twenty-four lactating donkeys were randomly assigned to one of the following three CP content diets: 15.3% (HP), 14.2% (MP), and 13.1% (LP) of dry matter, respectively. The experiment lasted for 10 weeks, with the first two weeks being used for adaptation. The results showed that milk yield and yields of protein, lactose, solid-not-fat, total solid, and contents of protein, total solid and milk urea nitrogen in the HP and MP groups were higher than the LP group. No significant changes were observed in dry-matter intake, contents of milk fat, lactose or solid-not-fat. The feed conversion ratio, milk protein synthesis efficiency, and the digestibility of dry matter, crude protein, ether extract, acid detergent fiber, neutral detergent fiber, calcium and phosphorous in the HP and MP groups were greater than the LP group. Serum total protein, albumin and urea nitrogen concentrations decreased, while concentrations of non-esterified fatty acids and β-hydroxybutyrate increased in the LP group compared with the HP and MP groups. In conclusion, the diet containing 14.2% CP supplied an adequate amount of protein for improving milk production in lactating donkeys, but milk production was not further increased by feeding the donkeys more than 14.2% CP.