Among the phospholipases A2 (PLA2S) are the group VI Ca2+-independent PLA2S (iPLA2s), and expression of multiple transcripts of iPLA2 in skeletal muscle has been reported. In the present study, phospholipase activity and sequential ATP and calmodulin affinity column chromatography analyses reveal that skeletal muscle iPLA2 exhibits properties characteristic of the iPLA 2β isoform. The phospholipase activity of iPLA2β has been demonstrated to participate in signal transduction, cell proliferation, and apoptosis. We report here that skeletal muscle from iPLA 2β-null mice, relative to wild-type muscle, exhibits a reduced capacity to oxidize palmitate but not palmitoyl-CoA or acetyl-CoA in the absence of changes in fatty acid transporters CD36 and CPT1 or β-hydroxyacyl-CoA dehydrogenase activity. Recently, purified iPLA2β was demonstrated to manifest a thioesterase activity which catalyzes hydrolysis of fatty acyl-CoAs. The liberated CoA-SH facilitates fatty acid transport into the mitochondria. In this regard, we find that fractions eluted from the ATP column and containing iPLA2β phospholipase activity also contained acyl-CoA thioesterase activity that was inhibited by the bromoenol lactone (BEL) suicide inhibitor of 1PLA2β. We further find that acyl-CoA thioesterase activity in skeletal muscle preparations from iPLA 2β-null mice is significantly reduced, relative to WT activity. These findings suggest that the absence of acyl-CoA thioesterase activity of 1PLA2β can lead to reduced fatty acyl-CoA generation and impair fatty acid oxidation in iPLA2β-null mice. Our findings therefore reveal a novel function of 1PLA2β, related not to its phospholipase activity but to its thioesterase activity, which contributes to optimal fatty acid oxidation in skeletal muscle. © 2008 American Chemical Society.