Previously, it was demonstrated that the novel proteasome inhibitor N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu‑1) possesses activity against chronic lymphocytic leukemia (CLL). In the present study, we attempted to assess whether this drug has a synergistic effect with fludarabine on the apoptosis of CLL cells. Annexin V/PI staining, mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) levels were examined by flow cytometry in short-term cell culture of blood cells from untreated newly diagnosed patients ex vivo. Expression of active caspase-3 and the Bcl-2/Bax ratio for determination of apoptosis were also investigated by flow cytometry and western blot analysis. Our results revealed that the ZGDHu-1 may induce the apoptosis of CLL cells through the mitochondrial pathway and its pro-apoptotic effect is CLL-specific, not affecting normal lymphocytes. Most importantly, a combination of ZGDHu-1 and a non-cytotoxic dose of fludarabine had a synergistic apoptotic effect. To some extent, caspase-3 activation may be involved in the mechanism of the ZGDHu-1 synergistic cytotoxic effect with fludarabine, as well as the cleavage of PARP, consequently leading to apoptosis. Notably, the rate of apoptosis caused by ZGDHu-1 alone or in combination with fludarabine was independent of prognostic markers of CLL disease such as ZAP-70 and CD38 expression or clinical Rai classification stage. In conclusion, ZGDHu-1 exhibited a significant synergistic effect with fludarabine to induce the apoptosis of CLL cells, which implies a possible clinical application.