L-Lysine is an essential amino acid for mammals, with two distinct tissue-specific degradative pathways: most tissues use the saccharopine pathway, while the central nervous system (CNS) primarily employs the pipecolate pathway. L-Pipecolate (L-PA), a metabolite in the latter pathway, may act as a neuromodulator to potentiate GABAergic inhibitory neurotransmission, whereas its downstream product L-R-aminoadipate has excitotoxic properties and reduces levels of the neuroprotective kynurenic acid. To investigate the role of L-PA and related metabolites in the CNS, we sought to develop specific inhibitors of L-pipecolate oxidase (L-PO), a primate flavoenzyme that catalyzes the oxidation of L-PA to ∆1-piperideine-6-carboxylate. Here we report that 4,5-dehydro-L-pipecolic acid (∆4,5-L-PA, 6) is the first example of a potent inhibitor of L-PO. Racemic 6 was synthesized and converted to homochiral ∆4,5-L-PA via an enzymatic-chemical procedure. Evaluation of its inhibitory effect on L-PO isolated from Rhesus monkey liver revealed time-dependent inhibition, with kinetic parameters KI = 130 µM and kinact = 2.0 min−1. The activity loss was largely reversible; however, incubation with high concentrations of 6 or for extended periods resulted in partial irreversible inactivation. In summary, ∆4,5-L-PA is the first potent mechanism-based inhibitor of L-PO. This compound serves as a novel pharmacological tool for studying the role of pipecolic acid and other lysine metabolites in the CNS and may provide leads for the development of new anticonvulsants.