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Rationale Ligusticum chuanxiong Hort is a well‐known herb medicine that has been widely prescribed to treat cardiovascular diseases in China for hundreds of years. Senkyunolide H (SNH) is one of the major bioactive ingredients extracted from L. chuanxiong, and it displayed neuroprotective effects. To fully understand its mechanism of action, the metabolism needs to be investigated. Methods In vitro studies were conducted by incubating SNH with rat and human hepatocytes, and the metabolites were identified and characterized using liquid chromatography in combination with hybrid quadrupole Orbitrap mass spectrometry (LC‐Orbitrap‐MS). The structures of the metabolites were proposed by accurate mass analysis of respective precursor ions, indicative product ions, and elemental compositions. Results Under the current conditions, a total of 10 metabolites were identified, and among these metabolites, M3 and M4 were the most abundant metabolites both in rat and human hepatocytes. Our results demonstrated that hydroxylation, hydration, glucuronidation, and GSH conjugation were the primary metabolic pathways of SNH. Conclusions The present study provides new information on the metabolism of SNH, which would help prospects of the disposition of SNH.