Lappaconitine (LAP), a non-addictive potent analgesic drug, is broadly used to treat cancer and postoperative pain in many countries, and it also has antibiotic activity against Pseudomonas aeruginosa and Salmonella Typhi. Despite its widespread usage and potential for expanded use, its metabolism was poorly investigated. In this work, the metabolic fate of LAP in liver microsomes of the rat and human was compared, and after oral administration, the metabolites in the rat were investigated using ultra high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UHPLC–Q/TOF-MS). As a result, a total of 51 metabolites were identified, including 48 metabolites that were reported here for the first time. Based on accurate MS/MS spectra and the known structure of LAP, the metabolites structures and their fragment ions were readily characterized. The biotransformations of LAP in vitro and in vivo were shown to involve hydroxylation, N-deacetylation, O-demethylation, N-deethylation, and hydrolysis. Furthermore, the results indicated a quantitative species difference in the metabolites for LAP between the rat and human. However, 16-DMLAP, DAL and 5′-OH-DAL were the main in vitro and in vivo metabolites. This work provides the LAP metabolite profiles in rat and human, which will help better understand the pharmacological and toxicological activities of LAP.