The new uses of the old drugs may reduce cost and shortens the production cycle of research and development, especially that approved by FDA for important clinical conditions. Alcohol abuse Disulfiram (DSF) has been proven safe and shows the promising anti-tumor effect in may preclinical studies. However, the potential side effects of DSF on tumor remain unknown. In this study, we explored the role of DSF in cancer cells and searched for the differential expressed protein after DSF treatment in the HeLa cells. To fully understand the mechanism of action of DSF with a systems perspective, we employed a quantitative proteomics strategy to systematically identify potential targets of DSF. In total, 201 proteins were dys-regulated significantly after DSF exposure, implying that they may be potential targets of DSF. Analysis of this data on a system level revealed major changes of proteins involved in diverse biological processes, including metabolic process and response to stimulus.