LPS treatment of macrophages induces TG accumulation, which is accentuated by TG-rich lipoproteins or FFA. We defined pathways altered during macrophage activation that contribute to TG accumulation. Glucose uptake increased with activation, accompanied by increased GLUT1. Oxidation of glucose markedly decreased, whereas incorporation of glucose-derived carbon into FA and sterols increased. Macrophage activation also increased uptake of FFA, associated with an increase in CD36. Oxidation of FA was markedly reduced, whereas the incorporation of FA into TGs increased, associated with increased GPAT3 and DGAT2. Additionally, macrophage activation decreased TG lipolysis; however, expression of ATGL or HSL was not altered. Macrophage activation altered gene expression similarly when incubated with exogenous FA or AcLDL. Whereas activation with ligands of TLR2 (zymosan), TLR3 (poly I:C), or TLR4 (LPS) induced alterations in macrophage gene expression, leading to TG accumulation, treatment of macrophages with cytokines had minimal effects. Thus, activation of TLRs leads to accumulation of TG in macrophages by multiple pathways that may have beneficial effects in host defense but could contribute to the accelerated atherosclerosis in chronic infections and inflammatory diseases.