The present study determined the role of DEP domain containing mTOR-interacting protein (DEPTOR) in the proliferation, apoptosis and chemosensitivity of RPMI-8226 multiple myeloma cells, using small hairpin RNA (shRNA) to knock down DEPTOR gene expression in vitro. DEPTOR mRNA and protein levels in RPMI-8226 cells treated with DEPTOR-specific shRNA were evaluated by reverse transcription–polymerase chain reaction and Western blotting. Expression of apoptosis-associated proteins (including cleaved caspase-3 and cleaved poly-ADP ribose polymerase [PARP]) and activation of the phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homologue 1 (AKT) signalling pathway were detected by Western blotting. Transfection of DEPTOR-specific shRNA successfully knocked down DEPTOR gene expression in transfected RPMI-8226 cells. These transfected cells, together with control RPMI-8226 cells, were treated with 20 µmol/l melphalan for 24 h. Knockdown of DEPTOR exacerbated melphalan-induced growth inhibition and apoptosis, increased levels of cleaved caspase-3 and cleaved PARP, and reduced levels of phosphor-AKT. Downregulation of DEPTOR inhibited proliferation and increased chemosensitivity to melphalan in human multiple myeloma RPMI-8226 cells via inhibiting the PI3K/AKT pathway.