Purpose: To investigate the effect of Peony seed oil (PO) on non-alcoholic fatty liver disease (NAFLD), and the underlying mechanism of action. Methods: A mouse model of NAFLD was established using high-fat diet (HFD) for 12 weeks. Serum levels of triacylglycerol (TG), total cholesterol (TC), glucose and free fatty acids (FFAs) were determined. Activities of aspartate aminotransferase (AST) and alanine amino transferase (ALT) were assayed in liver homogenates, while mRNA and protein expressions of GRP78, x-box binding protein 1 (XBP1) and phosphorylated inositol-requiring enzyme 1 (p-IRE1α) were assayed in liver tissue using real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting, respectively. Expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were also determined by qRT-PCR. Results: HFD diet led to significant increases in body weight as well as serum TG, TC, glucose and FFA levels. However, treatment with PO significantly reversed the effect of HFD on these parameters (p < 0.05). The mRNA expression levels of TNF- α, IL-6 and IL-1β were significantly higher in HFD control group than in normal control group, but were significantly reduced after PO treatment (p < 0.05). The protein expression levels of GRP78, XBP-1 and p-IRE1α significantly increased in the liver of NAFLD group, when compared with normal control rats, but were significantly downregulated by treatment with PO (p < 0.05). Conclusion: PO attenuates HFD-induced NAFLD via inactivation of endoplasmic reticulum stress (ERS) pathway. Thus, PO has potentials for the treatment of NAFLD but further studies are required to ascertain this.