The multifaceted roles of calcium-independent phospholipase A 2β (iPLA2β) in numerous cellular processes have been extensively examined through utilization of the iPLA2-selective inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Herein, we employed accurate mass/high resolution mass spectrometry to demonstrate that the active site serine (S465) and C651 of iPLA 2β are covalently cross-linked during incubations with BEL demonstrating their close spatial proximity. This cross-link results in macroscopic alterations in enzyme molecular geometry evidenced by anomalous migration of the cross-linked enzyme by SDS-PAGE. Molecular models of iPLA 2β constructed from the crystal structure of iPLA 2α (patatin) indicate that the distance between S465 and C651 is approximately 10 Å within the active site of iPLA2β. Kinetic analysis of the formation of the 75 kDa iPLA2β-BEL species with the (R) and (S) enantiomers of BEL demonstrated that the reaction of (S)-BEL with iPLA2β was more rapid than for (R)-BEL paralleling the enantioselectivity for the inhibition of catalysis by each inhibitor with iPLA2β. Moreover, we demonstrate that the previously identified selective acylation of iPLA2β by oleoyl-CoA occurs at C651 thereby indicating the importance of active site architecture for acylation of this enzyme. Collectively, these results identify C651 as a highly reactive nucleophilic residue within the active site of iPLA2β which is thioesterified by BEL, acylated by oleoyl-CoA, and located in close spatial proximity to the catalytic serine thereby providing important chemical insights on the mechanisms through which BEL inhibits iPLA2β and the topology of the active site.