Objective This study aims to investigate the main metabolites and metabolic pathways of Portulacatone B in rats, which is an alkaloid isolated from Portulaca oleracea L. Methods Portulacatone B was administered through the tail vein of the rat, and the orbital blood at 10 and 30 min and urine and feces within 24 h were collected. The metabolites and metabolic pathways in the rat were researched by ultra-high performance liquid chromatography-electrospray coupled with quadrupole-time of flight mass spectrometry (UHPLC-ESI-Q-TOF/MS). Results The research results of the metabolites and metabolic pathways of Portulacatone B showed that after administration through the tail vein of rats, 3 metabolites were found in the plasma sample, 2 metabolites in the urine sample, and one metabolite in the feces sample. The main metabolic pathways were found to be oxidation, hydrolysis, methylation, glucuronidation, and sulfonation. Conclusion Six metabolites were found in the rat's plasma, urine, and feces samples, and the metabolic pathways included oxidation, hydrolysis, methylation, glucuronidation, and sulfonation process.