Carbon tetrachloride (CCl4) is a common hepatotoxin used in experimental models to elicit liver injury. To identify the proteins involved in CCl4-induced hepatotoxicity, two-dimensional gel electrophoresis was employed followed by mass spectrometry - mass spectrometry (MS/MS) to study the differentially expressed proteins during CCl4 exposure in the Fischer 344 rat liver proteome for 5 weeks. Ten spots with notable changes between the Control and CCl4 groups were successfully identified. Among them, four proteins with significant up-regulation, namely calcium-binding protein 1, protein disulfide isomerase, mitochondrial aldehyde dehydrogenase precursor, and, glutathione-S-transferase mu1 and six proteins with significant down-regulation, namely catechol-O-methyltransferase, hemoglobin-alpha-2-chain, hemopexin precursor, methionine sulfoxide reductase A, catalase and carbonic anhydrase 3, were identified. The data indicates that CCl4 causes hepatotoxicity by depleting oxygen radical scavengers in the hepatocytes. In this rat model, we profiled hepatic proteome alterations in response to CCl4 intoxication. The findings should facilitate understanding of the mechanism of CCl4-induced liver injury.