Objective To evaluate the differences between three commonly used methods for establishing mouse models of premature ovarian insufficiency (POI), and to determine the method that is most accordant with the clinical features of POI. Methods Ninety-six ICR mice with regular estrous cycles were randomly divided into 4 groups: the control group (A), the tripterygium glycosides (TG) group (B), the 4-vinylcyclohexene diepoxide (VCD) group (C), and the D-(+)-galactose (D-gal) group (D). After intervention with the different chemicals, eight mice in each group were sacrificed, the serum sex hormone levels were measured, and the pathological changes of uterus and ovaries were examined. Results The level of E2 and the number of follicles differed significantly between mice in the model groups and those in the control group ( P <0. 01). And the estrous cycles in mice of the model groups were significantly irregular whereas those of the control group were regular. Conclusions Mouse models of POI can be successfully established with VCD via intraperitoneal injection. This method has the advantages of being an easier operation and having a higher success rate, as compared with the other two models method. It is the ideal one of the three establishing method and is suitable for popular use.