1,3-dioleoyl-2-palmitoylglycerol (OPO) and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) are two essential types of human milk fat substitutes (HMFS). Their unique fatty acid composition and distribution play a significant role in promoting infant health, making the reaction conversion and acyl migration critical factors for developing efficient preparation methods. Promoting the conversion of the substrate while simultaneously inhibiting acyl migration is crucial for obtaining the desired HMFS products. In this study, we comparatively investigated enzymatic acidolysis and transesterification for HMFS production and revealed enzymatic kinetics as well as acyl migration mechanism during the process. Acyl migration was observed through the lipase-catalyzed mechanism, and the associated free energy changes were analyzed using density functional theory (DFT). The presence of long-chain fatty acids in the synthesis system resulted in intermediates with higher relative free energy during acyl migration. Based on these findings, we propose a novel synthesis strategy consisting of multi-step transesterification and dry fractionation, leveraging the differences in freezing points to minimize acyl migration. The resulting OPO product contains 90.42% oleic acid specifically at the sn-1,3 positions, highlighting its potential application in infant formulas. This study presents a systematic investigation of the kinetics and mechanisms involved in lipase-mediated HMFS production, providing valuable insights for rational synthesis approaches.