Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic hepatic disorder. The naturally occurring phytosterol; β-sitosterol has antiobesogenic and anti-diabetic properties. The purpose of this study was to explore the role of β-sitosterol in preventing hepatic steatosis induced by a high-fat diet (HFD) in rats. In the current study, to induce NAFLD in the female Wister rats, an HFD was administered to them for 8 weeks. The pathogenic severity of steatosis in rats receiving an HFD diet was dramatically decreased by oral administration of β-sitosterol. After administering β-sitosterol to HFD-induced steatosis for three weeks, several oxidative stress-related markers were then assessed. We showed that β-sitosterol reduced steatosis and the serum levels of triglycerides, transaminases (ALT and AST) and inflammatory markers (IL-1β and iNOS) compared to HFD-fed rats. Additionally, β-sitosterol reduced endoplasmic reticulum stress by preventing the overexpression of inositol-requiring enzyme-1 (IRE-1α), X-box binding protein 1(sXBP1) and C/EBP homologous protein (CHOP) genes which, showing a function in the homeostatic regulation of protein folding. Also, it was found that the expression of the lipogenic factors; peroxisome proliferator-activated receptor (PPAR-α), sterol regulatory element binding protein (SREBP-1c) and carnitine palmitoyltransferase-1(CPT-1), which are involved in the regulation of the fatty acid oxidation process, may be regulated by β-sitosterol. It can be concluded that β-sitosterol may prevent NAFLD by reducing oxidative stress, endoplasmic reticulum stress and inflammatory responses, which supports the possibility of using β-sitosterol as an alternative therapy for NAFLD. Together, β-sitosterol may be an option for NAFLD prevention.