Microglial activation by oleate and palmitate differentially modulates brain inflammatory status. However, the metabolic reprogramming supporting these reactive phenotypes remains unknown. Employing real-time metabolic measurements and lipidomic analysis, we show that both fatty acids promote microglial oxidative metabolism, while lipopolysaccharide (LPS) enhances glycolytic rates. Interestingly, oleate treatment was followed by enrichment in storage lipids bound to polyunsaturated fatty acids (PUFA), in parallel with protection against oxidative imbalance. Palmitate, in turn, induced a distinct lipid distribution defined by PUFA linked to membrane phospholipids, which are more susceptible to lipid peroxidation and inflammatory signaling cascades. This distribution was mirrored by LPS treatment, which led to a strong pro-inflammatory phenotype in microglia. Thus, although both oleate and palmitate preserve mitochondrial function, a contrasting lipid distribution supports differences in fatty acid-induced neuroinflammation. These data reinforce the concept that reactive microglial profiles are achieved by stimulus-evoked remodeling in cell metabolism.