This study aims to investigate the hepatoprotective effect of date seeds oil (DSO) in mice subjected to a high cholesterol diet (HCD). First, the in vivo acute toxicity of DSO was tested. Then, the in vivo hepatotoxicity experiment was done on twenty-eight male mice randomly divided into four groups: a control group, an HCD group, a DSO-treated group, and a group receiving both HCD and DSO. Multiple biochemical analyses were conducted, including serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and C-reactive protein (CRP). Additionally, glutathione (GSH) and catalase (CAT) activity were analysed. Finally, a histopathological examination was done on liver tissues. The results demonstrated that DSO significantly reduced serum TG and cholesterol levels, ameliorated oxidative stress markers such as GSH and CAT, and exhibited a hepatoprotective effect, as evidence by attenuation of liver injury through the reduction of steatosis, as well it reduced the AST, ALT, and CRP levels. This suggests that DSO has potential as a therapeutic agent against HDC-induced hepatotoxicity.