Gallbladder cancer (GBC), highly aggressive form of cancer with an extremely poor prognosis, is the most common malignancy of the biliary tract. In this study, we investigated the effects of dioscin (DSN) on human GBC and the potential mechanisms underlying these effects. The results showed that DSN significantly inhibited GBC cell proliferation and migration. Moreover, DSN induced GBC cell apoptosis via mitochondrial dependent apoptotic signalling. Reactive oxygen species (ROS) and glutathione (GSH) levels were measured, and ROS scavengers completely inhibited DSN-induced apoptosis and migration, indicating that ROS play an essential role in GBC progression. Western blot analysis showed that AKT activity was significantly downregulated after DSN treatment, and that inhibition/ectopic expression of AKT enhanced/abolished DSN-induced apoptosis but not migration. Furthermore, we confirmed the relationship between ROS and the PI3K/AKT pathway and found that DSN induced apoptosis by regulating ROS-mediated PI3K/AKT signaling. Taken together, these findings indicate that DSN induces GBC apoptosis through inhibiting ROS-mediated PI3K/AKT signalling.