Abstract: The composition and regiodistribution of fatty acids influence the physical and the nutritional properties of milk fat.The fatty acid composition and the fatty acid composition at sn-2 position in mare and human milk were analyzed.The fatty acid composition at sn-2 position was measured by lipase hydrolysis after which 2-monoglycerides (2-MAG) were isolated by thin-layer chromatography (TLC) and were converted to fatty acid methyl esters, and then analyzed by gas-liquid chromatography.Results showed that palmitic (C16:0) , oleic (C18:1) and linoleic acid (C18:2) were 19.07%, 36.43%, 23.73% in human milk but they were 24.47%, 17.40%, 4.78% in mare milk.The saturated fatty acid fraction of human milk at sn-2 position made up approximately 59.41%, and was particularly rich in palmitic (C16:0) 48.36%, miristic acid (C14:0) 6.00%, but mare milk made up 71.46%, rich in palmitic (C16:0) 47.00%, miristic acid (C14:0) 12.48%, Distribution of fatty acid in mare milk similar to human milk whose distribution wasn't random which provided guidance about data and substrate for the research of human milk fat substitute.