Pyrrolizidine alkaloids (PAs) are considered to be one of the most hepatotoxic groups of compounds of plant origin and are present in about 3% of the world's flowering plants. Most PAs represent a considerable health hazard to both livestock and humans through the consumption of plants and PA-contaminated products such as milk, honey, herbal teas, and medicines. This study determined the differences in the in vivo pharmacokinetic behavior of senecionine (SEN), adonifoline (ADO), and their main metabolites in rats after intravenous administration and oral administration by ultraperformance liquid chromatography/electrospray ionization mass spectrometry. Upon intravenous administration and oral administration of SEN and ADO, significant differences in pharmacokinetics were observed, with the SEN and ADO being absorbed fast with lower bioavailability and being quickly metabolized to PA N-oxides and hydroxylation products of PAs or their N-oxides. It could be seen that the plasma concentration ratio of senecionine N-oxide (SEN-NO) to SEN (C (SEN-NO)/C (SEN)) was significantly larger than that for adonifoline N-oxide (ADO-NO) and ADO (C (ADO-NO)/C (ADO)) (P < 0.001) for both dosing routes in rats. The high N-oxygenation activity and extensive toxicity of SEN, compared with ADO, in rats raised the question of whether or not the higher metabolic rate of SEN in rats in vivo was related to its potent toxicity. The toxicity of SEN-NO and ADO-NO needs to be evaluated further and compared in vitro/in vivo. This study was most helpful for interpreting the metabolism of metabolic bioactivation and detoxication, and toxicity differences among SEN, ADO and other PAs.