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Ornithine aminotransferase (OAT) is overexpressed in hepatocellular carcinoma (HCC), and we previously showed that inactivation of OAT inhibits the growth of HCC. Recently, we found that (3<i>S</i>,4<i>S</i>)-3-amino-4-fluorocyclopentenecarboxylic acid (<b>5</b>) was a potent inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), proceeding by an enamine mechanism. Here we describe our investigations into the activity and mechanism of <b>5</b> as an inactivator of human OAT. We have found that <b>5</b> exhibits 10-fold less inactivation efficiency (<i>k</i>_inact/<i>K</i>_I) against <i>h</i>OAT than GABA-AT. A comprehensive mechanistic study was carried out to understand its inactivation mechanism with <i>h</i>OAT. p<i>K</i>_a and electrostatic potential calculations were performed to further support the notion that the α,β-unsaturated alkene of <b>5</b> is critical for enhancing acidity and nucleophilicity of the corresponding intermediates and ultimately responsible for the improved inactivation efficiency of <b>5</b> over the corresponding saturated analogue (<b>4</b>). Intact protein mass spectrometry and the crystal structure complex with <i>h</i>OAT provide evidence to conclude that <b>5</b> mainly inactivates <i>h</i>OAT through noncovalent interactions, and that, unlike with GABA-AT, covalent binding with <i>h</i>OAT is a minor component of the total inhibition which is unique relative to other monofluoro-substituted derivatives. Furthermore, based on the results of transient-state measurements and free energy calculations, it is suggested that the α,β-unsaturated carboxylate group of PLP-bound <b>5</b> may be directly involved in the inactivation cascade by forming an enolate intermediate. Overall, compound <b>5</b> exhibits unusual structural conversions which are catalyzed by specific residues within <i>h</i>OAT, ultimately leading to an enamine mechanism-based inactivation of <i>h</i>OAT through noncovalent interactions and covalent modification.