Glutamate (Glu), the key excitatory neurotransmitter in the central nervous system, is considered essential for brain functioning and has a vital role in learning and memory formation. earlier it was considered as a harmful agent but later found to be useful for many body functions. However, studies regarding the effects of free l-Glu administration on CNS function are limited. Therefore, current experiment is aimed to monitor the neurobiological effects of free l-Glu in male rats. l-Glu was orally administered to rats for 5-weeks and changes in behavioral performance were monitored. Thereafter, brain and hippocampus were collected for oxidative and neurochemical analysis. Results showed that chronic supplementation of free l-Glu enhanced locomotor performance and cognitive function of animals which may be attributed to the improved antioxidant status and cholinergic, monoaminergic and glutamatergic neurotransmission in brain and hippocampus. current results showed that chronic supplementation of l-Glu affects the animal behaviour and brain functioning via improving the neurochemical and redox system of brain. free l-Glu could be a useful therapeutic agent to combat neurological disturbances however this requires further targeted studies. Central nervous system (CNS) modulates behaviour and brain functioning with the help of multiple neurotransmitters. One of the important neurotransmitters is Glutamate (Glu) 1 which is a nonessential amino acid (AA) present abundantly in the body 2. It is involved in a variety of body functions either directly or by converting into other products 3. Evidence shows the importance of Glu in facilitating protein synthesis 4 , intestinal nutrition, cell signalling, gene expression modulation, immune responses 5 , regulating blood glucose level, removing excess ammonia 6 , disposal of excess or waste nitrogen 7 as well as in intermediary metabolism 2 as an important energy fuel 5. Other than this, it is also a precursor of various biologically active components like glutathione (GSH), GABA, purine and pyrimidine nucleotides, polyamines, poly-glutamated folate cofactors or certain AAs like glutamine, alanine, aspartate, proline, arginine, citrulline 2-5,8. Glu is present in various foods either as naturally occurring free Glu (vegetable, seafood, milk, meat, cheese) or as a by-product of hydrolysed protein (used in seasoning) or as a salt; MSG (monosodium glutamate), a food additive or flavouring agent in canned foods, dry mixes, sauces, and soups 9 .