In response to inflammatory activation by pathogens, macrophages accumulate triglycerides in intracellular lipid droplets. The mechanisms underlying triglyceride accumulation and its exact role in the inflammatory response of macrophages are not fully understood. Here, we aim to further elucidate the mechanism and function of triglyceride accumulation in the inflammatory response of activated macrophages. Lipopolysaccharide (LPS)-mediated activation markedly increased triglyceride accumulation in macrophages. This increase could be attributed to up-regulation of the hypoxia-inducible lipid droplet–associated (HILPDA) protein, which down-regulated adipose triglyceride lipase (ATGL) protein levels, in turn leading to decreased ATGL-mediated triglyceride hydrolysis. The reduction in ATGL-mediated lipolysis attenuated the inflammatory response in macrophages after ex vivo and in vitro activation, and was accompanied by decreased production of prostaglandin-E2 (PGE2) and interleukin-6 (IL-6). Overall, we provide evidence that LPS-mediated activation of macrophages suppresses lipolysis via induction of HILPDA, thereby reducing the availability of proinflammatory lipid precursors and suppressing the production of PGE2 and IL-6.