Human milk fat (HMF) is a perfect nutritional source that includes all the required ingredients which are necessary for the growth of infants up to 6 months. Although its composition may differ among mothers or during lactation stage, its unique triacylglycerol (TAG) structure remains constant which is characterized by the presence of palmitic acid (PA) at the sn-2 position. Previous reports provided convincing information of higher PA and calcium absorption and efficient use of dietary energy when at this specific position in the TAG moiety than when PA is at the sn-1,3 positions. During the design of structured lipids (SLs) intended for infant nutrition, this unique property is taken into consideration. Human milk fat substitutes (HMFS) enriched with important fatty acids such as omega-3 and omega-6 fatty acids are intended to better mimic the functions of HMF as well as provide associated health benefits. The use of microencapsulation technology and novel technologies such as ultrasound technology in conjunction with SL production and enzyme-catalyzed reactions are evolving and ongoing issues in infant formula production. Therefore, further studies should be directed towards new process improvements in order to increase the functional properties and oxidative stabilities of HMFS. Novel technologies in lipid biotechnology related to HMFS preparation should also be explored.