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• Chemical composition and bioactivity of Acer truncatum seeds were investigated. • Oil yield, fatty acids and nervonic acid content varied greatly among locations. • Fifteen compounds from the seed residue were putatively identified in A. truncatum. • Seed residue showed cytotoxicity in human cancer cells and anti-AChE activity. • Prioritize Inner Mongolia samples for further study and product development. Acer truncatum , an ornamental, medicinal, and edible plant, is a tree endemic to China and now widely cultivated throughout Asia, North America, and Europe. It has been developed as an industrial crop from its traditional use as a substitute for tea. The oil from its seeds is rich in unsaturated fatty acids (FA), especially in nervonic acid (NA). However, studies on the chemical components and bioactivities of the seeds are limited, and comparative studies of seeds collected from different locations are even more limited. Therefore, A. truncatum seeds were collected from 12 different locations in China to investigate the chemical composition, cytotoxicity, and acetylcholinesterase (AChE) inhibition of A. truncatum seed oil (ATO) and Acer seed residue extracts (ASR). The oil yields varied about five-fold among different locations from 7.27% to 35.05%. Nine fatty acids were identified and relatively quantified, and myristic acid, linoleic acid, oleic acid, and a NA isomer [C24:1(15 E)] were the dominant fatty acids. The NA isomer has not been reported previously in A. truncatum , and NA levels ranged from 0.66 to 14.86 mg/μL of ATO. This is the first GC–MS study to focus on the FA differences of A. truncatum collected from different locations. Using UPLC-QTOF-MS analysis, 15 compounds were putatively identified from ASR, including N -acetyl-D-tryptophan, nonanedioic acid, 10 E -heptadecenoic acid, pregnanolone, phosphatidylglycerol (34:2), and phosphatidylinositol (34:1), which have not been reported in Acer prior to this study. The flavonoids miscanthoside and isovitexin/vitexine were also newly identified in A. truncatum. Furthermore, five samples each of ATO and ASR were selected to estimate the cytotoxic activities against four human tumor cell lines (A-549, SMMC-7721, MCF-7, and SW-480) and the AChE inhibitory activity. All ASR were cytotoxic on all tested cells and exhibited higher inhibitory activity than ATO at 100 μg/mL. ASR exhibited stronger cytotoxicity on SMMC-7721 with IC 50 from 29.46 to 52.55 μg/mL. Similarly, the ASR exhibited higher AChE inhibitory activity than the ATO. All ASR significantly inhibited AChE activity, and the inhibition rates were close to 90% at 20 μg/mL. The IC 50 values for the ASR ranged from 0.124 to 0.232 μg/mL. Overall, the results of this study indicate that ATO is abundant in FAs and NA, but the contents of ATO varies by location. ASR are rich in bioactive components with cytotoxic and AChE inhibitory activities. These findings suggest that ATO and ASR could be a promising resource in the nutraceutical and pharmaceutical fields. [ABSTRACT FROM AUTHOR]