L-proline is an amino acid with a unique cyclic structure, involvement in various physiological processes, such as protein synthesis, collagen production, and neurotransmission. This review explores the complex roles of proline in the central nervous system (CNS), where it contributes to both excitatory and inhibitory neurotransmission. Additionally, L-proline has distinct metabolic functions attributed to its structural properties. The concentration-dependent effects of L-proline indicate its importance in CNS function, with potential implications for health and disease. Studies in animal models suggest that L-proline influences cognitive function and behavior, with dysregulated levels linked to learning and memory deficits. Furthermore, this review addresses the neuropathological consequences of hyperprolinemia, a metabolic disorder marked by elevated L-proline levels in the CNS and examines the potential role of L-proline in neurological and psychiatric disorders. In sum, this work provides a comprehensive perspective on the neurobiological importance of L-proline, underscoring its involvement in neurotransmission, behavioral modulation, and disease pathology.