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Objective To observe the effects of the proteasome inhibitor bortezomib (BTZ) on the apoptosis of murine alloreactive T cells in vitro. Methods In a one-way mixed lymphocyte culture system, the T lymphocytes from C57BL/6 mice were co-cultured with the splenocytes of BALB/c mice for 24 h. After treatment of the cells with different concentrations of bortezomib for 12 h, the changes in the cell proliferation were assessed using CCK-8 assay. The responses of the co-cultured cells to different culture times and a 12 h treatment with 0.01 μmol/L bortezomib were examined by detecting the changes in the cell apoptosis, cell cycle and expression of the activation-associated markers CD25 and CD69 using flow cytometry; the expression of cleaved caspase-3 and cyclin D1 was detected using Western blotting, and the levels of interleukin-4 (IL-4), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in the supernatant were measured using enzyme-linked immunosorbent assay (ELISA). Results Compared with the control cells, the bortezomib-treated T cells showed an obviously decreased proliferative activity. Treatment with 0.01 μmol/L bortezomib significantly increased the percentage of apoptotic T cells and enhanced the expression of cleaved caspase-3 in positive correlation with the co-culture time prior to bortezomib treatment (P < 0.05). The proportion of T cells at G1 phase increased and the expression of cyclin D1 decreased significantly in bortezomib-treated cells (P < 0.05), which also showed down-regulated expressions of CD25 and CD69 and lowered levels of IL-4, TNF-α and IFN-γ in the supernatant (P < 0.05). Conclusion Bortezomib can inhibit the proliferation of murine alloreactive T cells in vitro, induce apoptosis and reduce cytokine production in the activated T cells.