This study aimed to investigate potential new target(s)/mechanism(s) for the palmitoylethanolamide (PEA) analogue, adelmidrol, and its role in an in vitro model of contact allergic dermatitis. Freshly isolated canine keratinocytes, human keratinocyte (HaCaT) cells and human embryonic kidney (HEK)-293 cells, wild-type or transfected with cDNA encoding for N-acylethanolamine-hydrolysing acid amidase (NAAA), were treated with adelmidrol or azelaic acid, and the concentrations of endocannabinoids (anandamide and 2-arachidonoylglycerol) and related mediators (PEA and oleoylethanolamide) were measured. The mRNA expression of PEA catabolic enzymes (NAAA and fatty acid amide hydrolase, FAAH), and biosynthetic enzymes (N-acyl phosphatidylethanolamine-specific phospholipase D, NAPE-PLD) and glycerophosphodiester phosphodiesterase 1, was also measured. Brain or HEK-293 cell membrane fractions were used to assess the ability of adelmidrol to inhibit FAAH and NAAA activity, respectively. HaCaT cells were stimulated with polyinosinic-polycytidylic acid and the release of the pro-inflammatory chemokine, monocyte chemotactic protein-2 (MCP-2), was measured in the presence of adelmidrol. Adelmidrol increased PEA concentrations in canine keratinocytes and in the other cellular systems studied. It did not inhibit the activity of PEA catabolic enzymes, although it reduced their mRNA expression in some cell types. Adelmidrol modulated the expression of PEA biosynthetic enzyme, NAPE-PLD, in HaCaT cells, and inhibited the release of the pro-inflammatory chemokine MCP-2 from stimulated HaCaT cells. This study demonstrates for the first time an 'entourage effect' of adelmidrol on PEA concentrations in keratinocytes and suggests that this effect might mediate, at least in part, the anti-inflammatory effects of this compound in veterinary practice.