Abstract Multiple myeloma is a fatal plasma-cell malignancy that evolves mainly in the bone marrow. Melphalan is widely used to treat patients with Multiple Myeloma, however its mechanism of action remains poorly documented. Thus, we studied the effect of this drug on the molecules of the Bcl-2 family. We showed that melphalan induces a drastic down-regulation of the anti-apoptotic proteins Mcl-1L and Bcl-xL and the pro-apoptotic BimEL in human melphalan-sensitive myeloma cells while the most potent pro-apoptotic Bim isoforms, L and S, are less affected. Interestingly, the disappearance of Mcl-1L and BimEL is associated with the generation of the respective cleaved forms, described as pro-apoptotic. We determined that the cleaved forms were generated by caspase cleavage since the addition of zVADfmk inhibited their formation. Indeed, we observed that caspase 3 activation occurred as early as Mcl-1 cleavage. Recently, we demonstrated that Mcl-1, the major anti-apoptotic molecule for myeloma cells, neutralizes the pro-apoptotic Bim molecule through endogenous complex formation and therefore, preventing the activation of death effectors. In this study, we demonstrate that melphalan disrupts the Mcl-1/Bim complex while the Bcl-2/Bim complex is not affected. In turn, the disappearance of full length Mcl-1 allows the release of Bim isoforms, particularly L and S, which can exert their pro-apoptotic function. In addition, we demonstrate both Bax activation and cytochrome c release, supporting the role of free Bim as a major initiator of the mitochondrial apoptotic cascade. Thus, we propose that in myeloma cells melphalan induces the disruption of Mcl-1/Bim complex resulting in the release of Bim from Mcl-1 sequestration and therefore leading to the initiation of the apoptotic process. Moreover, the cleaved 26kDa pro-apoptotic Mcl-1 and the 19kDa and 12kDa of Bim, generated during melphalan treatment could contribute to the amplification loop of apoptosis. MM remains a fatal disease which develops multi-drug resistance, including melphalan. A potential therapeutic approach could be the disruption of the Mcl-1/Bim complexes by the BH3 small molecules that might target those complexes leading to the release of sequestered Bim.