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.