Update information. Milk fat globule membrane (MFGM) proteins, which are recognized to play a variety of biological roles; nevertheless, their composition and interspecies complexity remain unknown. This study aimed to identify and quantify proteins extracted from MFGM-enriched fractions of bovine, goat and camel milk by a label free proteomics approach. We identified 1579 proteins, substantially increasing the number of MFGM proteins identified for these species. The results of functional analysis showed that the identified MFGM proteins had similar functional annotations, with cellular processes, intracellular anatomical structures and binding being the predominant GO annotations. And most of the proteins were involved in KEGG pathways such as protein processing of ribosomes and endoplasmic reticulum. Subsequently, multiple statistical methods were used to compare the differences in MFGM protein quantification among different species. The results of principal component analysis showed that bovine, goat and camel milk MFGM proteins were significantly different, among which CSN1S1, XDH, LTF and PI4KA played an important role in taxonomic identification. Meanwhile the results of hierarchical clustering showed that the differences (p<0.05) between bovine and goat milk were smaller than those of camel milk. These new data deepen the understanding of the protein composition of MFGM and its possible physiological roles, providing directions for the production of specific functional milk proteins.