Human milk fat substitutes (HMFSs), rich in palmitic acid (P) at the sn-2 position of triacylglycerol (TAG), were prepared from lard via Novozym435(®)- and Lipozyme RM-IM(®)-mediated two-step reactions. First, 2-palmitoyl monoacylglycerol (2-P-MAG, 90% purity) was prepared via Novozym435(®)mediated ethanolysis of lard. Then, 2-P-MAG, oleic acid (O), linoleic acid (L), and lard were dissolved in hexane and subjected to a Lipozyme RM-IM(®)-mediated reaction for HMFS preparation. The effect of the amount of 2-P-MAG and fatty acids (O and L) in HMFS preparation were investigated. Under the optimum reaction conditions: 7 mmol of lard, 3.0 mmol of 2-P-MAG, 5.2 mmol of O, 3.5 mmol of L, 10 mL of hexane, 10 wt% of Lipozyme RM-IMR(®) (against the total weight of substrates), 550 rpm, 37°C and 6 h, a HMFS with total fatty acid composition and P content at the sn-2 position of TAG similar to that of human milk fat was prepared. In the same way, a HMFS having polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (PUFA-HMFS) was prepared. The HMFS and PUFA-HMFS prepared in this study, as well as fats extracted from commercially available powdered milk for infants (FPM) were evaluated for their oxidation stability by an auto-oxidation test. The test showed HMFS and PUFA-HMFS to possess greater oxidation stability than that the FPMs. These results indicated that the HMFS and PUFA-HMFS prepared in this study have value as potential ingredients for powdered milk.