AbstractThe metabolism of amyloid β‐protein precursor (APP) is regulated by various cytoplasmic and/or membrane‐associated proteins, some of which are involved in the regulation of intracellular membrane trafficking. We found that a protein containing Asp–His–His–Cys (DHHC) domain, alcadein and APP interacting DHHC protein (AID)/DHHC‐12, strongly inhibited APP metabolism, including amyloid β‐protein (Aβ) generation. In cells expressing AID/DHHC‐12, APP was tethered in the Golgi, and APP‐containing vesicles disappeared from the cytoplasm. Although DHHC domain‐containing proteins are involved in protein palmitoylation, a AID/DHHC‐12 mutant of which the enzyme activity was impaired by replacing the DHHC sequence with Ala–Ala–His–Ser (AAHS) made no detectable difference in the generation and trafficking of APP‐containing vesicles in the cytoplasm or the metabolism of APP. Furthermore, the mutant AID/DHHC‐12 significantly increased non‐amyloidogenic α‐cleavage of APP along with activation of a disintegrin and metalloproteinase 17, a major α‐secretase, suggesting that protein palmitoylation involved in the regulation of α‐secretase activity. AID/DHHC‐12 can modify APP metabolism, including Aβ generation in multiple ways by regulating the generation and/or trafficking of APP‐containing vesicles from the Golgi and their entry into the late secretary pathway in an enzymatic activity‐independent manner, and the α‐cleavage of APP in the enzymatic activity‐dependent manner.