Objective: This study aimed to investigate the main metabolites and metabolic pathways of oleraisoquinoline in rats, a new alkaloid isolated from Portulaca oleracea L., and test its antioxidation and anticholinesterase effects. Methods: Ultra-high-performance liquid chromatography-electrospray coupled with quadrupole time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF/MS) was applied to study the metabolism of oleraisoquinoline. Furthermore, 1,1-diphenyl-2-picrylhydrazyl assay and modified Ellman’s method were used to test the antioxidation and anticholinesterase effects of oleraisoquinoline, re-spectively. Results: The metabolism results of oleraisoquinoline showed, after its administration through the tail vein of rats, 4 metabolites in the plasma samples, 17 metabolites in the urine sample, and 2 metabolites in the feces sample. The main metabolic pathways were hydrolyzation, oxidation, hydroxylation, sulfonation, glucuronidation, acetylation, and methylation. Additionally, IC50 values of antioxidant and anticholinesterase activities were 13.819 ± 0.005 μM and 10.551 ± 0.069 μM, respectively. Conclusion: 21 metabolites were found in the rat’s plasma, urine, and feces samples, and the metabolic pathways included hydrolyzation, oxidation, hydroxylation, sulfonation, glucuronidation, acetylation, and methylation; among them, sulfonation was the main metabolic reaction. Mean-while, oleraisoquinoline also showed extremely good antioxidant and anticholinesterase activities. © 2023 Bentham Science Publishers.