Human milk oligosaccharides (HMOs) have attracted considerable attention due to their unique role in boosting infant health. Among the HMOs, lacto-N-tetraose (LNT) is a significant constituent associated with various health benefits, such as prebiotic effects, antiadhesive antimicrobials, antiviral protection, and immune modulators. LNT has received a “Generally Recognized as Safe” status by the American Food and Drug Administration and was approved as a food ingredient for infant formula. However, the limited availability of LNT poses a major challenge for its application in food and medicine. In this review, we first explored the physiological functions of LNT. Next, we describe several synthesis methods for production of LNT, including chemical, enzymatic, and cell factory approaches, and summarize the pivotal research results. Finally, challenges and opportunities for the large-scale synthesis of LNT were discussed.