The major vault protein (MVP) is the main constituent of the vault ribonucleoprotein particle. Although the role in multidrug resistance has been suggested, the physiological function of vaults remains unclear. It has been reported that MVP levels were elevated after treatment with the DNA-damaging agents such as adriamycin (ADR). The aim of this study was to investigate molecular basis for the induction of MVP by ADM, and to investigate the potential role of MVP in drug resistance. Expression levels of both MVP protein and MVP mRNA were increased by ADM. ADM could also enhance MVP promoter activity. Introduction of siRNA against Sp-1 in SW620 cells attenuated the expression of MVP induced by ADM. Down-regulation of MVP expression by MVP RNAi in SW620 cells increased the sensitivity of the cells to ADM. Furthermore, ADM enhanced the activation of caspase 3 and caspase 8 in the MVP knock-down cells, but not in control cells. Our data demonstrate that exposure of cancer cells to DNA-damaging agents induces MVP that might have an important role in the resistance to chemotherapeutic agents.