Diosbulbin B (DB) is the main hepatotoxic compound in Airpotato yam, which is traditionally used for treating thyroid disease and cancer in China, and its hepatotoxic mechanism still remains unclear. This study aims to investigate its hepatotoxic mechanism by focusing on regulating microRNA (miRNA). DB induced hepatotoxicity both in vivo and in vitro. Results of miRNA chip analysis showed that the expression of eleven miRNAs was up-regulated and twelve miRNAs was down-regulated in livers from DB-treated mice. The altered expression of seven miRNAs was further validated by using real-time polymerase chain reaction (RT-PCR) assay. DB induced G2/M arrest in L-02/cytochrome P450 3A4 (CYP3A4) cells in both concentration- and time-dependent manner. A total of eleven predicted target genes was related with cell cycle regulation of those above seven miRNAs, among which the mRNA and protein expression of cyclin-dependent kinase 1 (CDK1) decreased both in vivo and in vitro. Both miR-378a-5p and miR-186-3p have binding sites in the 3′-untranslated region (UTR) of CDK1. With the use of CDK1 3′-UTR luciferase reporter assay, miR-378a-5p and miR-186-3p was found to down-regulate the luciferase activity. The mimics of miR-378a-5p or miR-186-3p reduced CDK1 expression in L-02/CYP3A4 cells, but their inhibitors reversed the decreased CDK1 expression induced by DB. Moreover, overexpression of miR-186-3p inhibitor reversed the G2/M cell cycle arrest induced by DB in L-02/CYP3A4 cells. Taken together, our results showed that DB induced hepatotoxicity by inducing G2/M cell cycle arrest in hepatocytes via miR-186-3p or miR-378a-5p-mediated the reduced CDK1 expression.