Human milk fat (HMF) is a natural complex lipid possessing a unique fatty acids composition and distribution. In order to mimic HMF, human milk fat substitutes (HMFSs) have been produced via the enzymatic or chemical modifications of natural lipids. HMFSs have attracted increasing attention as functional lipids owing to their beneficial effects on infant ’s growth and development. It is very difficult to prepare HMFSs that will exactly match HMF in all lipid compositions. However, it is possible to produce HMFSs by enzymatic reactions that closely resemble HMF. There are two approaches for HMFSs synthesis. The first approach is performed in one step by acidolysis of one triacylglycerol with free fatty acids or interesterification between two triacylglycerols or between one triacylglycerol and fatty acids methyl or ethyl esters. The second approach can be accomplished in two steps through hydrolysis and re-esterification with the acyl groups to be incorporated.