FTY720 (fingolimod), a synthetic sphingosine 1-phosphate (S1P) analogue, is a drug employed for treating relapsing-remitting multiple sclerosis in diverse age groups. With the ability to cross the blood-brain barrier, FTY720 gradually accumulates within lipid-rich areas of the central nervous system (CNS) by integrating into phospholipid membranes. Consequently, FTY720 can enhance cell membrane fluidity, influencing glial cells and neuronal populations involved in behaviour regulation. This study investigated the impact of chronic FTY720 administration (3.3 μg/ml in ad libitum drinking water for 3 weeks) on behaviour and CNS lipid composition in adolescent C57BL6/J male mice during a crucial stage of brain maturation, specifically focusing on the hippocampus—a critical brain region for learning, memory, and sensory-emotional processing. While FTY720 altered anxiety-like behaviours, memory and spatial learning remained unaffected, as evidenced by the Open Field and Morris Water Maze tests, respectively. Using mass spectrometry-based lipidomics, our study revealed FTY720-induced modifications in the hippocampal lipidome, showcasing an increase in the fatty acid chain length of hydroxy-phosphatidylcholine (PCOH) lipids. Furthermore, FTY720 led to a decrease in monounsaturated fatty acids (MUFAs) and an increase in polyunsaturated fatty acids (PUFAs) of PCOH lipids. A total of 99 lipid species were upregulated, while only 3 were downregulated in the mouse hippocampus following FTY720 exposure. This study presents a wide overview of the lipid classes and lipid species that are altered in the hippocampus following chronic FTY720 exposure and provides novel insight into cellular and molecular mechanisms underlying FTY720’s therapeutic or adverse effects in the CNS.