The prognosis of unresectable advanced gastric cancer has improved over the last decade due to advances in chemotherapy. However, molecular targeting in gastric cancer therapy has been poorly established and the 5‑year survival rate is still <10%. The proteasome plays a pivotal role in the regulation of cell proliferation, apoptosis and differentiation in a variety of tumor cells. Bortezomib, a selective inhibitor of the proteasome, has prominent effects against several tumor types, including multiple myeloma. We examined the anti-tumor effects of bortezomib on gastric cancer cells in vitro and in subcutaneously transplanted nude mice. We demonstrated that among seven types of gastric cancer cells examined, treatment with bortezomib induced both apoptotic and anti-proliferative effects, resulting in a reduction in cell survival rates. The induction of apoptosis was observed to be dependent on the inhibition of nuclear factor κB (NF-κB) activation and the subsequent production of reactive oxygen species (ROS) and c-Jun N-terminal kinase (JNK) activation. Interestingly, we observed that those cells with high levels of NF-κB activity were resistant to bortezomib treatment. Additionally, we demonstrated that the activation of the extracellular signal-regulated kinase (ERK1/2) was inhibited following bortezomib treatment, which may contribute to its anti-proliferative effects. We also observed anti‑tumor effects of bortezomib in vivo. Bortezomib is a potential novel molecular targeting drug for the treatment of unresectable advanced gastric cancer.