Clioquinol, a chelator and ionophore of copper/zinc, was extensively used as an amebicide to treat indigestion and diarrhea in the mid-1900s. However, it was withdrawn from the market in Japan because its use was epidemiologically linked to an increase in the incidence of subacute myelo-optic neuropathy (SMON). Yet, the pathogenesis of SMON has not been fully elucidated. A reporter assay revealed that clioquinol activated metal response element-dependent transcription in human neuroblastoma SH-SY5Y cells. Clioquinol significantly increased the cellular level of zinc within 1 h, possibly due to its ionophore effects. On the other hand, clioquinol significantly increased the cellular level of copper within 24 h. Clioquinol induced the oxidation of the copper chaperone antioxidant 1 (ATOX1), suggesting its inactivation and inhibition of copper transport. The secretion of dopamine-β-hydroxylase (DBH) and lysyl oxidase, both of which are copper-dependent enzymes, was inhibited by clioquinol, along with decreased noradrenaline levels. In fact, disruption of the ATOX1 gene suppressed the secretion of DBH. Taken together, the disturbance of cellular copper transport by the inactivation of ATOX1 may be one of the mechanisms involved in clioquinol-induced neurotoxicity in SMON.