D-Galactosamine (D-GaIN) is a highly selective hepatotoxin that causes liver injury similar to human viral hepatitis via depletion of uridine nucleotides, which subsequently diminishes synthesis of RNA and proteins. The aim of this study was to investigate the role of selenium, ascorbic acid, beta-carotene, and alpha-tocopherol on D-GaIN-induced liver injury of rats by morphological and immunohistochemical means. In this study, Sprague-Dawley female rats were divided into four groups. Group I consists of rats injected physiologic saline solution intraperitoneally. Group II consists of rats given selenium (0.2 mg/kg per day), ascorbic acid (100 mg/kg per day), beta-carotene (15 mg/kg per day), and alpha-tocopherol (100 mg/kg per day) for 3 days via gavage method. Group III consists of the single dose of D-GaIN (500 mg/kg)-injected animals. Group IV are the D-GaIN-injected animals given the same antioxidant combination. In situ terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL) assay was applied to determine apoptosis for paraffin sections of the liver samples. Moreover, caspase-3 and proliferating cell nuclear antigen antibody were applied for paraffin sections. In the group given D-GaIN, apoptotic cells with TUNEL assays and caspase-3 activity, which are liver injury markers induced by D-GaIN, the hepatocyte proliferation with cell proliferation assay increased. However, selenium and other three antioxidants combination clearly suppressed an increase in apoptotic cells with TUNEL assay and caspase-3 activity. In addition, it suppressed D-GaIN-induced cell proliferation in the liver. As a result, these results indicate that selenium and three naturally occurring antioxidants shows a protective effect against liver injury induced by D-GaIN. These results suggest that supplementation with the combination of selenium, ascorbic acid, beta-carotene, and alpha-tocopherol may help prevent the development of liver injury.