Black soybean seed (BSS) is a traditional edible and medical food. The cotyledons of BSS are either yellow or green, but that difference's effect on functional and bioactivities are not clear. In the present study, the concentrations of the main functional components, including anthocyanins, isoflavones, fatty acids, protein, and polysaccharides, and in vitro antioxidant activities of yellow-cotyledon and green-cotyledon BSS (YBS and GBS, respectively) were compared. The anti-fatigue activities of the ethanol extract of BSS (EEB) were evaluated using mouse swimming endurance tests. Additionally, a metabolomics comparison between YBS and GBS was done using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) on high doses of EEB-treated mouse plasma. The results indicated that YBS showed significantly higher contents of anthocyanin and protein, and stronger antioxidant activity and anti-fatigue activity than GBS; while GBS showed higher levels of isoflavone, stearic acid, and polysaccharide. By infusing the feed with EEB, the swimming time of mice increased in a dose-dependent manner. The metabolomics results suggested that the anti-fatigue effect of EEB correlated well with serotonin and melatonin biosynthesis in vivo. All of the above results showed that BSS has anti-fatigue potential and might be used as a potential source of functional food ingredients. This study was financially supported by the National Natural Science Foundation of China (Grant No. 31971853) and the China Postdoctoral Science Foundation (Grant No. 2014 M560724 & 2017T100707). Peer reviewed