Mitochondrial DNA (mtDNA) mutations are often observed in various cancer types. Although correlation between mitochondrial dysfunction and cancer malignancy have been demonstrated by several researchers, more research is required to elucidate the molecular mechanisms underlying accelerated tumor development and progression due to mitochondrial mutations. We generated an mtDNA-depleted cell line, ρ0, through long-term ethidium bromide treatment to define the molecular mechanism of tumor malignancy induced by mitochondrial dysfunction. Mitochondrial dysfunction in ρ0 cells reduced drug-induced cell death and decreased expression of pro-apoptotic protein including p53. p53 expression was reduced on activation of the nuclear factor-κB that depended on elevated levels of free calcium in HCT116/ρ0 cells. Taken together, these data provide a novel mechanism for tumor development and drug resistance due to mitochondrial dysfunction.