Abstract Palmitoylethanolamide (PEA) is an endocannabinoid-like compound and the parent molecule of the aliamide family, a group of fatty acid amides able to act through the down-regulation of mast cell degranulation. PEA has been proven to exert both analgesic and anti-inflammatory activity, and recent studies have shown its ability in reducing clinical symptoms of inflammatory skin diseases, both in humans and in animals. Although its pharmacological efficacy is well known, the mechanism of action of this family of compounds is still unclear. To better understand the cellular effects of aliamides in dogs, canine mast cells freshly isolated from skin biopsies were incubated with IgE-rich serum and were challenged with anti-canine IgE. Histamine, prostaglandin D 2 (PGD 2) and tumour necrosis factor-alpha (TNFα) release was measured in the presence and absence of increasing concentrations of PEA, ranging from 10 −8 M to 10 −5 M. Histamine, PGD 2 and TNFα release, immunologically induced by canine anti-IgE, were significantly inhibited in the presence of PEA. The maximum inhibitory effect on histamine release was observed at 3 × 10 −6 M PEA concentration achieving an inhibition of 54.3 ± 5.2%. PGD 2 release was significantly inhibited at 10 −5 M and 10 −6 M PEA concentrations with 25.5 ± 10.2% and 14.6 ± 5.6% of inhibition, respectively. Finally, PEA inhibited TNFα release to 29.2 ± 2.0% and 22.1 ± 7.2%, at concentrations of 10 −5 M and 3 × 10 −6 M, respectively. The results obtained in the present study showed the ability of the aliamide PEA to down-modulate skin mast cell activation. Therefore, our findings suggest that the beneficial effect of PEA, observed in inflammation and pain clinical studies, could be due, at least in part, to its ability to inhibit the release of both preformed and newly synthesised mast cell mediators.