Although human milk fat substitute (HMFS) has been wildly applied in infant formula (IF), there are still some differences in fat composition between human milk and IF, which is extremely attributed to the phospholipid composition. We therefore designed a human milk phospholipid substitute (HMPS), and investigated its nutritional outcomes on brain development using C57BL/6J mice. Results showed that HMPS improved mice’s cognitive behavior and dendritic development compared to soybean phospholipids and no-phospholipid diets. Based on the lipidomics, we revealed that these beneficial outcomes were potentially associated with the increased biosynthesis of N-acylethanolamines, phosphatidylethanolamine (PE), and ether PE, and the decreased metabolism of diacylglycerol and hexosylceramide in brain. It was further found in Kyoto Encyclopedia of Genes and Genomes analysis that glycerophospholipid metabolic pathway participated in the improvement of HMPS on mice’s neurodevelopment. In conclusion, this study demonstrated that HMPS improves mice’s neurodevelopment, and the glycerophospholipid metabolic pathway plays a major role in this beneficial result, and provided evidence for future application of HMPS in commercial IF.