1,3-Dioleoyl-2-palmitoylglycerol (OPO)-rich human milk fat substitute (HMFS) was synthesized from tripalmitin (PPP)-rich fraction and oleic ethyl ester by a lipase-catalyzed interesterification. Response surface methodology (RSM) was employed to optimize the presence of palmitic acid at sn-2 position (Y_1, %) and of oleic acid at sn-1,3 (Y_2, %), with the reaction factors as substrate molar ratio of PPP-rich fraction to oleic ethyl ester (X_1, 1:4, 1:5 and 1:6), reaction temperature (X_2, 50, 55 and 60℃), and time (X_3, 3, 7.5 and 12 h). The optimal conditions for HMFS synthesis were predicted at the reaction combination of 55℃, 3 h and 1:6 substrate ratio. HMFS re-synthesized under the same conditions displayed 70.70% palmitic acid at the sn-2 position and 69.58% oleic acid at the sn-1,3 position. Reaction product was predominantly (90.35%) triacylglycerol (TAG) was observed in which the major TAG species, OPO, comprised 31.24%.