Calcium-independent phospholipase A2β (iPLA 2β) participates in numerous diverse cellular processes, such as arachidonic acid release, insulin secretion, calcium signaling, and apoptosis. Herein, we demonstrate the highly selective iPLA2β-catalyzed hydrolysis of saturated long-chain fatty acyl-CoAs (palmitoyl-CoA ≈ myristoyl-CoA ≫ stearoyl-CoA ≫ oleoyl-CoA ≈ arachidonoyl-CoA) present either as monomers in solution or guests in host membrane bilayers. Site-directed mutagenesis of the iPLA2β catalytic serine (S465A) completely abolished acyl-CoA thioesterase activity, demonstrating that Ser-465 catalyzes both phospholipid and acyl-CoA hydrolysis. Remarkably, incubation of iPLA2β with oleoyl-CoA, but not other long-chain acyl-CoAs, resulted in robust stoichiometric covalent acylation of the enzyme. Moreover, S465A mutagenesis or pre-treatment of wild-type iPLA2β with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one unexpectedly increased acylation of the enzyme, indicating the presence of a second reactive nucleophilic residue that participates in the formation of the fatty acyl-iPLA2β adduct. Radiolabeling of intact Sf9 cells expressing iPLA2β with [3H]oleic acid demonstrated oleoylation of the membrane-associated enzyme. Partial trypsinolysis of oleoylated iPLA 2β and matrix-assisted laser desorption ionization mass spectrometry analysis localized the acylation site to a hydrophobic 25-kDa fragment (residues ∼400-600) spanning the active site to the calmodulin binding domain. Intriguingly, calmodulin-Ca2+ blocked acylation of iPLA2β by oleoyl-CoA. Remarkably, the addition of low micromolar concentrations (5 μM) of oleoyl-CoA resulted in reversal of calmodulin-mediated inhibition of iPLA2β phospholipase A 2 activity. These results collectively identify the molecular species-specific acyl-CoA thioesterase activity of iPLA2β, demonstrate the presence of a second active site that mediates iPLA 2β autoacylation, and identify long-chain acyl-CoAs as potential candidates mediating calcium influx factor activity.