<p>Gastric cancer is one of the common malignant tumours in clinical practice with poor prognosis and high mortality. Icariside II is a single compound extracted from the traditional Chinese medicine <i>Epimedium brevicornu</i>&#xa0;Maxim, and it is also the main active ingredient of <i>Epimedium brevicornu</i>&#xa0;Maxim that exerts pharmacological effects. Studies have shown that Icariside II has anti-tumour activity, but its mechanism of action on gastric cancer cells is unclear. This study aims to analyze the impact of Icariside II on gastric cancer cells as well as on xenograft tumor models of gastric cancer, and to examine the potential molecular regulatory pathways. GES-1, a normal gastric cell line, and gastric cancer cell lines AGS and MGC803 were cultured to investigate the cytotoxic effects of Icariside II using the methylthiazolyldiphenyl-tetrazolium (MTT). Flow cytometry (FCM) was employed to measure the impact of Icariside II on the apoptosis levels of gastric cancer cells, while western blot analysis was used to examine the expression of apoptosis-related proteins and the Wnt/β-catenin signaling pathway. Subsequently, a xenograft tumor model was established and treated with Icariside II to observe changes in tumor volume and weight in the model mice. Finally, alterations in the expression of the Wnt/β-catenin signaling pathway were assessed through immunofluorescence (IF) and immunohistochemistry (IHC). The results showed that Icariside II had faint significant toxic effect on GES-1 cells, and was able to inhibit the proliferative activity and promote apoptosis of the gastric cancer cells. Moreover, Icariside II was able to inhibit the growth of gastric cancer in nude mice subcutaneous transplantation tumor. In addition, both in vivo and in vitro results indicated that Icariside II inhibited the activation of the Wnt/β-catenin signaling pathway. Icariside II inhibited tumorigenicity of gastric cancer by suppressing the Wnt/β-catenin signaling.</p>