AbstractHuman milk fat contains 20–25% palmitic acid (16∶0) and 30–35% oleic acid (18∶1). More than 60% of the plamitic acid occurs at the sn‐2 position of the glycerol backbone. Palm oil is a rich source of both palmitic and oleic acids. The structured lipid 1,3‐dioleyl‐2‐palmitoylglycerol (OPO) is an important ingredient in infant formula. OPO was synthesized from palm oil by a three‐step method. In the first step, low‐temperature fractionation was applied to palm oil FA, yielding a palmitic acid‐rich fraction (87.8%) and an oleic acid‐rich fraction (96%). The palmitic acid content was further increased to 98.3% by transforming palmitic acid into ethyl palmitate. In the second step, esterification of ethyl palmitate and glycerol catalyzed by lipase Novozym 435 under vacuum (40 mm Hg) was employed for the synthesis of tripalmitin. Finally, OPO was obtained by the reaction of tripalmitin. Finally, OPO was obtained by the reaction of tripalmitin with oleic acid catalyzed by Lipase IM 60. In this final step, the TAG content in the product acylglycerol mixture was 97%, and 66.1% oleic acid was incorporated into TAG. Analysis of the FA composition at the sn‐2 position of TAG showed 90.7 mol% of palmitic acid and 9.3 mol% of oleic acid. OPO content in the product TAG was ca. 74 mol%. Thus, an efficient method was developed for the synthesis of OPO from palm oil.