Recently, donkey milk has been indicated as a nutraceutical food thanks to some bioactive compounds interesting in the human diet; these substances are the lipids, which are characterized by the ability in conditioning indirectly or directly the intestinal environment and immunity, taking part in the prevention and treatment of some pathologies. With the aim to compare some nutritional properties of human and donkey milk, the triacylglycerol (TAG) composition and the positional isomers of donkey milk samples were analyzed by HPLC-APCI-MS on reversed phase and silver ion columns. The technique allowed the identification of 72 TAGs in the samples analyzed. Similarities and differences among the TAGs fraction of human and donkey milk can be easily determined by HPLC analysis of the lipid fraction. Donkey milk presents TAGs with partition number (PN) values starting from 30 up to 50. In human milk, the short-chain fatty acids (FAs) are not well-represented and the PN values range between 36 and 52. Other significant differences are among TAGs containing polyunsaturated fatty acids (PUFA). In fact, donkey milk presents a larger number and amount of ω3 and ω6 FAs than human milk, which contains only significant amount of ω6 FA (linoleic). Both donkey and human milk present the saturated FA preferably on the sn-2 position. Data testify a certain degree of similarity of these products in relation to their digestibility and confirm the increasing interest toward donkey milk as an alternative food for hypoallergenic diet in humans.