Background: The mechanism of a water-soluble extract of safflower on chronic alcoholic liver injury is still unclear. Objective: To study the mechanism of water extract of safflower on chronic alcoholic liver injury. Materials and Methods: The rats were separated in a random manner into five cohorts, namely, the normal control group (NC), alcoholic fatty liver disease model (ALD), ALD rat model treated with the water-soluble extract of safflower (30 g/kg), ALD rat model treated with the water-soluble extraction of safflower (40 g/kg), and ALD rat model with the water-soluble extract of safflower (50 g/kg). Rats in each group were anaesthetized with 2% isoflurane and euthanized by cervical dislocation after 1, 7, 14, 21, and 28 days treatment. Proteomic analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG analysis), immunohistochemistry (IHC), and western blot were employed for comparing the variations across model cohort and control cohort at the tissue level, protein level, and molecular level, respectively, and for probing potential prophylactic role/mechanisms for safflower extract on alcoholic liver injury. Results: KEGG analysis found that the differentially expressed genes in the alcoholic liver injury model were closely associated with alcoholism, inflammation, and serotonergic synapses. Furthermore, with the administration of water-soluble extract of safflower, these hepatic function indices and liver steatosis lesions were alleviated in both time and dose-dependent manners. Importantly, IHC analysis discovered that proteins disulfide isomerase A3 (PDIA3) and Trk-fused gene (TFG) were highly increased in the liver of the rats. Furthermore, the water-soluble extract of safflower alleviated liver oxidative injury and lipid peroxidation, via the upregulation of proteins such as 26S proteasome non-ATPase regulatory subunit 2 (PSMD2), cytochrome P450 2C6 (CYP2C6V1), and cytochrome P450 2C11 (CYP2C11), anddownregulation of arachidonate 12-lipoxygenase and 12S-type (ALOX12) protein expression. Conclusion: The water-soluble extract of safflower exhibited potential protective effects against lipid peroxidation and ethanol-driven oxidative damage within the liver via the upregulation of expression of proteins PSMD2, CYP2C6V1, and CYP2C11and downregulation of the expression of ALOX12, PDIA3, and TFG proteins.