Our previous study showed that the novel proteasome inhibitor NPI-0052 induces apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib (Velcade, Takeda, Boston, MA, USA) therapies. In vivo studies using human MM-xenografts demonstrated that NPI-0052 is well tolerated, prolongs survival, and reduces tumour recurrence. These preclinical studies provided the basis for an ongoing phase-1 clinical trial of NPI-0052 in relapsed/refractory MM patients. Here we performed pharmacodynamic (PD) studies of NPI-0052 using human MM xenograft murine model. Our results showed that NPI-0052: (i) rapidly left the vascular compartment in an active form after intravenous (i.v.) administration, (ii) inhibited 20S proteasome chymotrypsin-like (CT-L, beta5), trypsin-like (T-L, beta2), and caspase-like (C-L, beta1) activities in extra-vascular tumours, packed whole blood (PWB), lung, liver, spleen, and kidney, but not brain and (iii) triggered a more sustained (>24 h) proteasome inhibition in tumours and PWB than in other organs (<24 h). Tissue distribution analysis of radiolabeled compound (3H-NPI-0052) in mice demonstrated that NPI-0052 left the vascular space and entered organs as the parent compound. Importantly, treatment of MM.1S-bearing mice with NPI-0052 showed reduced tumour growth without significant toxicity, which was associated with prolonged inhibition of proteasome activity in tumours and PWB but not normal tissues.