Human milk fat substitute (HMFS) was prepared by Lipozyme RM IM-catalyzed acidolysis of lard and fatty acids obtained from palm kernel oil, tea seed oil and soybean oil, in a solvent-free system. The effects of substrate mass ratio, Lipozyme RM IM load and reaction time on the total fatty acids composition and sn-2 fatty acids composition were investigated. To optimize the reaction conditions, an orthogonal design was selected with three levels and three factors. Substrate mass ratio, Lipozyme RM IM load and reaction time were the factors employed. Under the given reaction temperature of 60 A degrees C, the optimal reaction conditions were, 1/2 mass ratio of lard/fatty acids blend, 7% Lipozyme RM IM load and 1-h reaction time. By the "deducting score" principle, the produced HMFS in comparison with the reported HMFS possessed highest degree of similarity with local HMF from Guangzhou mothers. The results showed that it was possible to produce HMFS through the scale-up enzymatic acidolysis.