Secretory phospholipase A 2 s (sPLA 2) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although group X (GX) sPLA 2 is recognized as the most potent mammalian sPLA 2 in vitro, its precise physiological function (s) remains unclear. We recently reported that GX sPLA 2 suppresses activation of the liver X receptor in macrophages, resulting in reduced expression of liver X receptor-responsive genes including ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and a consequent decrease in cellular cholesterol efflux and increase in cellular cholesterol content (Shridas et al. 2010. Arterioscler. Thromb. Vasc. Biol. 30: 2014–2021). In this study, we provide evidence that GX sPLA 2 modulates macrophage inflammatory responses by altering cellular cholesterol homeostasis. Transgenic expression or exogenous addition of GX sPLA 2 resulted in a significantly higher induction of TNF-α, IL-6, and cyclooxygenase-2 in J774 macrophage-like cells in response to LPS. This effect required GX sPLA 2 catalytic activity, and was abolished in macrophages that lack either TLR4 or MyD88. The hypersensitivity to LPS in cells overexpressing GX sPLA 2 was reversed when cellular free cholesterol was normalized using cyclodextrin. Consistent with results from gain-of-function studies, peritoneal macrophages from GX sPLA 2-deficient mice exhibited a significantly dampened response to LPS. Plasma concentrations of inflammatory cytokines were significantly lower in GX sPLA 2-deficient mice compared with wild-type mice after LPS administration. Thus, GX sPLA 2 amplifies signaling through TLR4 by a mechanism that is dependent on its catalytic activity. Our data indicate this effect is mediated through alterations in plasma membrane free cholesterol and lipid raft content.