Obesity leads to the appearance of an inflammatory process, which can be initiated even with a moderate weight gain. Palmitoylethanolamide (PEA) is an endogenous lipid, secreted by human adipocytes, that possesses numerous anti-inflammatory properties. The main purpose of this study was to investigate the anti-inflammatory effect of PEA on human adipocytes, as well as in a murine model. The production of tumor necrosis factor–α (TNF-α) by lipopolysaccharide (LPS)-treated human subcutaneous adipocytes in primary culture and CF-1 mice was investigated by enzyme-linked immunosorbent assay. The effects of PEA on adipocyte TNF-α secretion were explored as well as some suspected PEA anti-inflammatory pathways: nuclear factor–κB (NF-κB) pathway, peroxisome proliferator-activated receptor–α (PPAR-α) gene expression, and TNF-α-converting enzyme (TACE) activity. The effects of PEA on the TNF-α serum concentration in intraperitoneally LPS-treated mice were also studied. We demonstrate that the LPS induced secretion of TNF-α by human adipocytes is inhibited by PEA. This action is neither linked to a reduction in TNF-α gene transcription nor to the inhibition of TACE activity. Moreover, PPAR-α is not implicated in this anti-inflammatory activity. Lastly, PEA exhibits a wide-reaching anti-inflammatory action as the molecule is able to completely inhibit the strong increase in TNF-α levels in the serum of mice treated with high doses of LPS. In view of its virtual lack of toxicity, PEA might become a potentially interesting candidate molecule in the prevention of obesity-associated insulin resistance.