A comprehensive glycosylation profile of donkey lactoferrin, isolated by ion exchange chromatography from an individual milk sample, was obtained by means of chymotryptic digestion, TiO2 and HILIC enrichment, reversed-phase high performance liquid chromatography, electrospray mass spectrometry, and high collision dissociation fragmentation. The results obtained allowed the identification of 26 different glycan structures, including high mannose, complex and hybrid N-glycans, linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476. Altogether, the N-glycan structures determined revealed that in donkey milk lactoferrin most of the N-glycans identified are neutral complex/hybrid. Actually, 10 neutral non-fucosylated complex/hybrid N-glycans and 4 neutral fucosylated complex/hybrid N-glycans were found. In addition, 2 high mannose N-glycans, 4 sialylated fucosylated complex/hybrid N-glycans and 6 sialylated non-fucosylatedN-glycans, one of which containing N-glycolylneuramin acid (Neu5Gc), were found. A comparison of the glycosylation profile of donkey milk lactoferrin with respect to that of human, bovine and goat milk lactoferrin is reported.