The cadmium ion (Cd²⁺) is a highly toxic metal ion; however, its hepatic toxic effects are not very well characterized in a systematic manner. In this study, a zebrafish liver cell line, ZFL was used as a model to investigate the mechanism of Cd²⁺-induced cytotoxicity on hepatocytes. The intracellular level of reactive oxygen species decreased following the administration of Cd²⁺; antioxidant levels and related enzyme activities and gene expression were detected, showing that the toxic effects of Cd²⁺ might not be coupled to oxidative stress. To understand the cytotoxic effects of Cd²⁺ on ZFL cells after Cd²⁺ exposure, a total of 77 differentially expressed proteins were detected by two-dimensional gel electrophoresis; 43 of them were further identified by MALDI-TOF-MS. The proteins that responded to Cd²⁺ in ZFL cells were related to stress response, transporters, regulation of transcription, redox homeostasis, or different signaling pathways, with half of these proteins having metal ion binding capabilities.