The purpose of this study was to investigate the antitumor effects and drug interactions of the proteasome inhibitor Bortezomib against high-risk myelodysplastic syndrome (MDS) cells in vitro and in vivo. The high-risk MDS-derived MUTZ-1 cell line and bone marrow mononuclear cells from primary high-risk MDS patients were used to examine antitumor activity and drug interactions for Bortezomib. Apoptotic proteins, including caspase and Bcl-2 family members, as well as the protein FLIP, were studied. Phosphoinositide 3-kinase (PI3K)/Akt and MAPK signaling pathways were also examined. The PI3K inhibitor LY294002 was used to examine the involvement of the PI3K/Akt signaling pathway in the induction of apoptosis. Cytarabine (AraC) and daunorubicin (DNR) were used to test for synergistic effects between Bortezomib and chemotherapeutic agents. SCID mice xenografted with MUTZ-1 cells were used for in vivo study. We found that Bortezomib could induce growth arrest and apoptosis in high-risk MDS cells in vitro and in vivo. The mechanisms were related to decreased activation of the PI3K/Akt survival signaling pathway, but not the MAPK pathway, and involved inhibition of the NF-κB activity and downregulation of the Bcl-2/Bax and FLIPL/FLIPS ratios, triggering the activation of caspase cascades. This phenomenon was inhibited by the PI3K inhibitor LY294002. Bortezomib also acted synergistically with the chemotherapeutic agents AraC and DNR, which are associated with the inhibition of NF-κB activity. Our results demonstrate that Bortezomib can induce growth arrest and apoptosis of high-risk MDS cells and had a synergistic effect with two chemotherapeutic agents. Our findings provide new insights for the treatment of high-risk MDS, using either Bortezomib alone, or in combination with conventional antineoplastic agents.