Gastric cancer, due to its high incidence rate, is the second leading cause of cancer-related mortality worldwide. Chemotherapy is an important component of the multimodal treatment for gastric cancer; however, a significant impediment to successful treatment is multidrug resistance (MDR) in patients with gastric cancer. In the present study, the protein profiles of the MDR cell line, SGC7901/DDP, and its parental cell line, SGC7901, were comparatively analyzed through an iTRAQ-based quantitative proteomics technique. The protein tetraspanin-8 (TSPAN8) was found to be highly expressed in the SGC7901/DDP cells. To examine the role of TSPAN8 in the MDR of SGC7901/DDP cells, we increased cell sensitivity to drugs by increasing apoptosis. Additionally, the silencing of TSPAN8 downregulated Wnt pathway activity, β-catenin expression and β-catenin transfer to the nucleus. TSPAN8 was found to bind to NOTCH2, facilitating its mediation of the Wnt/β-catenin pathway by regulating β-catenin expression. Overall, the suppression of TSPAN8 expression may prove to be a promising strategy which may aid in the development of novel gastric cancer therapeutic drugs.