Copper (Cu) is the active center of some enzymes because of its redox-active property, although that property could have harmful effects. Because of this, cells have strict regulation/detoxification systems for this metal. In this study, multi-disciplinary approaches, such as speciation and elemental imaging of Cu, were applied to reveal the detoxification mechanisms for Cu in cells bearing a defect in Cu-regulating genes. Although Cu concentration in metallothionein (MT)-knockout cells was increased by the knockdown of the Cu chaperone, Atox1, the concentrations of the Cu influx pump, Ctr1, and another Cu chaperone, Ccs, were paradoxically increased; namely, the cells responded to the Cu deficiency despite the fact that cellular Cu concentration was actually increased. Cu imaging showed that the elevated Cu was compartmentalized in cytoplasmic vesicles. Together, the results point to the novel roles of MT and cytoplasmic vesicles in the detoxification of Cu in mammalian cells.