Palmitoleic acid (PM, 16:1n‐7) has anti‐inflammatory properties that could be linked to higher expression of PPARα, an inhibitor of NFκB. Macrophages play a major role in the pathogenesis of chronic inflammation, however, the effects of PM on macrophages are underexplored. Thus, we aimed to investigate the effects of PM in activated macrophages as well the role of PPARα. Primary macrophages were isolated from C57BL/6 wild type (WT) and PPARα knockout (KO) mice, cultured under standard conditions and exposed to lipopolysaccharides LPS (2.5 μg/ml) and PM 600 μmol/L conjugated with albumin for 24 hours. The stimulation with LPS increased the production of interleukin (IL)‐6 and IL‐1β while PM decreased the production of IL‐6 in WT macrophages. In KO macrophages, LPS increased the production of tumour necrosis factor (TNF)‐α and IL‐6 and PM decreased the production of TNFα. The expression of inflammatory markers such NFκB and IL1β were increased by LPS and decreased by PM in both WT and KO macrophages. PM reduced the expression of MyD88 and caspase‐1 in KO macrophages, and the expression of TLR4 and HIF‐1α in both WT and KO macrophages, although LPS had no effect. CD86, an inflammatory macrophage marker, was reduced by PM independently of genotype. PM increased PPARγ and reduced PPARβ gene expression in macrophages of both genotypes, and increased ACOX‐1 expression in KO macrophages. In conclusion, PM promotes anti‐inflammatory effects in macrophages exposed to LPS through inhibition of inflammasome pathway, which was independent of PPARα, PPARϒ and AMPK, thus the molecular mechanisms of anti‐inflammatory response caused by PM is still unclear.