AbstractA structured lipid (SL) with a high amount of sn‐2 palmitic acid was synthesized from anhydrous milkfat and was then enriched with docosahexaenoic (DHA) and arachidonic (ARA) acids using an immobilized lipase. Three different methods were compared including physical blending, enzymatic interesterification, and enzymatic acidolysis. Products were compared with respect to differences in fatty acid profiles, reaction times, antioxidant contents, oxidative stability, melting and crystallization profiles, and reaction yields. The acidolysis method was the least suitable for the synthesis of desired product because of a low reaction yield, low incorporation of DHA, low oxidative stability, and the extra processing steps required. The physical blending and interesterification methods were suitable, but the interesterification product (IE‐SL) had higher amounts of ARA at the sn‐2 position. The IE‐SL contained total ARA and DHA of 0.63 and 0.50 mol%, and 0.55 and 0.46 mol% at the sn‐2 position, respectively. The IE‐SL also contained 44.97 mol% sn‐2 palmitic acid. The reaction yield for the IE‐SL was 91.84 %, and its melting completion and crystallization onset temperatures were 43.1 and 27.1 °C, respectively. This SL might be totally or partially used in commercial fat blends for infant formula.