Diterpenoid lactones (DLs), a group of furan-containing compounds found in Dioscorea bulbifera L. (DB), have been reported to be associated with hepatotoxicity. Different hepatotoxicities of these DLs have been observed in vitro, but reasonable explanations for the differential hepatotoxicity have not been provided. Herein, the present study aimed to confirm the potential factors that contribute to varied hepatotoxicity of four representative DLs (diosbulbins A, B, C, F). In vitro toxic effects were evaluated in various cell models and the interactions between DLs and CYP3A4 at the atomic level were simulated by molecular docking. Results showed that DLs exhibited varied cytotoxicities, and that CYP3A4 played a modulatory role in this process. Moreover, structural variation may cause different affinities between DLs and CYP3A4, which was positively correlated with the observation of cytotoxicity. In addition, analysis of the glutathione (GSH) conjugates indicated that reactive intermediates were formed by metabolic oxidation that occurred on the furan moiety of DLs, whereas, GSH consumption analysis reflected the consistency between the reactive metabolites and the hepatotoxicity. Collectively, our findings illustrated that the metabolic regulation played a crucial role in generating the varied hepatotoxicity of DLs.