The simplest and the most cost-effective way of human milk fat substitute (HMFS) production is formulating of suitable vegetable oils at proper ratios. To do this, the D-optimal mixture design was used to optimize the HMFS formulation. The design included 25 formulations made from refined palm olein (35-55%), soybean oil (5-25%), olive oil (5-20%), virgin coconut oil (5-15%), and fish oil (0-10%). Samples were produced in laboratory and characterized in terms of fatty acid and triacylglycerol (TAG) compositions, free fatty acid content, peroxide value, iodine value, and oxidative stability index (OSI). HMFS samples were also compared with Codex Alimentarius (CA) and Iran National Standards Organization (INSO) standards. Each characteristic of HMFS samples was then expressed as a function of ingredient ratio using regression models. Finally, using numerical optimization, four optimized blends (PB 1 -PB 4 ) were selected, made in the laboratory (HMFS 1 -HMFS 4 ), characterized, and compared with CA and INSO standards. The properties of all the optimized blends (except the palmitic acid content of HMFS 2 and the monounsaturated fatty acid [MUFA] content of HMFS 3 ) met the standards. HMFS 4 showed the highest OSI in Rancimat and the lowest oxidation rate in Schaal oven tests. POL (19.53-21.73%), PPO (20.77-21.73%), .46%) were the main (totally about 60%) TAG species found in HMFS samples. In conclusion, the HMFS 4 formula (55% palm olein, 13.5% soybean oil, 16% refined olive oil, 15% virgin coconut oil, and 0.5% fish oil) was suggested as the best formula for HMFS production.Keywords Human milk fat substitutes Á Optimization Á Vegetable oils Á Fish oil Á D-optimal mixture design J Am Oil Chem Soc (2019) 96: 555-569.