Update information. Cryptococcus neoformans is an invasive human fungal pathogen, which causes pneumonia and meningoencephalitis in both healthy and immunodeficient individuals, resulting 181,100 deaths each year. Studies have demonstrated that copper detoxification machineries play critical functions in modulating C. neoformans fitness and pathogenicity in the host lung tissue. Pulmonary C. neoformans infection provokes the generation of toxic copper bombardment, which in return activate detoxification process in fungal cells. However, the molecular mechanism on how Cu inhibits C. neoformans in proliferation remains unclear. Here, using Cu detoxification gene knockout strains we demonstrated that exogenous Cu ions inhibit cell growth of the metallothionein mutant, which was significantly rescue when supplementing with the ROS scavenger, N-acetylcysteine. To future characterize the molecular mechanism of C. neoformans response to Cu toxicity, we employ iTRAQ with LC-MS/MS analysis, in the presence of exogenous Cu or NAC. Our data showed that an increased Cu level repressed expression of factors involved in protein translation but activates expression of important players in ubiquitin-degradation process. We proposed that the downregulation of protein synthesis and the upregulation of protein degradation were the main strategy of Cu toxicity. The metallothionein mutant showed a higher ubiquitination level under Cu treatment. In addition, MG132 could partially restore the Cu toxicity effect on metallothionein mutant. These results shed new light on the Cu antifungal mechanisms from proteomic profile.