The enhancement of the anticancer activity by disulfiram (DSF) chelated with copper (DSF/Cu²⁺) has been investigated recently, while the underlying molecular mechanisms still need to be fully elucidated. Chloride channel-3 (ClC-3) is over-expressed in a variety of cancers and involves multiple tumor biological events. However, whether the over-expression of ClC-3 in tumor cells affects the sensitivity of anti-tumor drugs remains unclear. Here, we showed that the involvement of ClC-3 chloride channel in the selective cytotoxicity of DSF/Cu²⁺ in the poorly-differentiated nasopharyngeal carcinoma. The EC₅₀ of DSF alone and DSF/Cu²⁺ in activating the Cl⁻ channel were 95.36 μM and 0.31 μM in the CNE-2Z cells, respectively. DSF/Cu²⁺ exhibited a positive correlation between the induction of the Cl⁻ currents and the inhibition of cell proliferation. DSF/Cu²⁺ increased the ClC-3 protein expression and induced the cell apoptosis. Cl⁻ channel blockers, NPPB and DIDS, and ClC-3 siRNA partially inhibited the cell apoptosis, and depleted the Cl⁻ currents induced by DSF/Cu²⁺ in CNE-2Z cells. However, these effects could not be observed in the normal nasopharyngeal epithelium NP69-SV40 T cells. In vivo, the transplanted human nasopharyngeal carcinoma tumors size in the DSF/Cu²⁺ group decreased about 73.2% of those in the solvent control group. The chloride blockers partially inhibited the antitumor action of DSF/Cu²⁺. These data demonstrated that the selective cytotoxicity of DSF/Cu²⁺ may relate to its selective activation of ClC-3 Cl⁻ channel pathways in CNE-2Z cells. ClC-3 Cl⁻ channel can be viewed as a new and promising target for the treatment of nasopharyngeal carcinoma.