The anticancer activity of disulfiram (DS) is copper(ii) (Cu)-dependent. This study investigated the anticancer mechanisms of DS/Cu using in vitro cytotoxicity and metabolic kinetic analysis. Our study indicates that DS/Cu targets cancer cells by the combination of two types of actions: (1) instant killing executed by DS/Cu reaction generated reactive oxygen species; (2) delayed cytotoxicity introduced by the end product, DDC-Cu. Nanoencapsulation of DS might shed light on repositioning of DS into cancer treatment.