The linoleic acid derivative DCP-LA serves as a selective activator of PKCε. The present study aimed at understanding the mechanism underlying PKCε-regulated α7 nicotinic acetylcholine (ACh) receptor trafficking. DCP-LA specifically bound to N-ethylmaleimide-sensitive factor (NSF) in lysates from the rat brain. PKCε, activated by DCP-LA, phosphorylated NSF at the serine residues, but not the threonine residues, in differentiated PC-12 cells. DCP-LA enhanced an association of NSF/α7 nicotinic ACh receptor in PC-12 cells, and the effect was cancelled by the PKC inhibitor GF109203X. DCP-LA increased cell surface localization of α7 nicotinic ACh receptor in PC-12 cells, which was abolished by GF1090203X or knocking-down NSF. DCP-LA also increased cell surface localization of α7 nicotinic ACh receptor in rat hippocampal slices, and the effect was suppressed by GF109203X, the vesicular transport inhibitor latrunculin B, or the vesicular exocytosis inhibitor botulinum toxin A. Collectively, the results of the present study show that PKCε, activated by DCP-LA directly binding to NSF, phosphorylates NSF at the serine residues, facilitates an association of NSF/α7 nicotinic ACh receptor, and stimulates NSF-dependent vesicular exocytosis of α7 nicotinic ACh receptor.