Bufadienolides are a type of natural steroids possessing potent antitumor activities. In this study, 4 arenobufagin structure-related bufadienolides were selected for the incubation experiments with human liver microsomes (HLMs) to investigate the structure-metabolism relationships. Arenobufagin (3) is the parent nucleus structure they shared. Both argentinogenin (1) and 4β-hydroxyl-5α-argentinogenin (2) hold the double bond between C9 and C11. Each of 4β-hydroxyl-5α-argentinogenin (2) and 4β-hydroxyl-5α-arenobufagin (4) has an additional 4β-hydroxyl group (4β-OH). Among them 4β-hydroxyl-5α-argentinogenin (2) was a new compound. 14 major metabolites were found and analyzed by UHPLC-ESI-Orbitrap mass spectrometer. Reduction reaction was the main reaction type of argentinogenin (1) and 4β-hydroxyl-5α-argentinogenin (2). Besides, hydroxylation reaction also occurred at argentinogenin (1). For 4β-hydroxyl-5α-arenobufagin (4), the major metabolic type was dehydrogenation. Arenobufagin (3) held the most reaction types that included the above forms. Through comparison, the double bond between C9 and C11 in compound 1 and 2 was considered more active than the carbonyl group at C-12 in the reduction reaction. 4β-OH, in compound 2 and 4, was deemed to block the hydroxylation reaction by steric effects. In the meanwhile, it also resulted in a significant decrease in the metabolic rate.