Background: Liver injury due to ischemia/reperfusion (I/R) is a major challenge during liver surgery. Objectives: Effects of paeonol pretreatment on partial hepatic I/R injury were assessed in this study. Methods: Male Wister rats were allocated into sham, vehicle-I/R, and paeonol-I/R groups. Paeonol, 100 mg/kg/day, was administered by gavage. After 10 days, rats of I/R groups were subjected to partial I/R of the liver. Blood samples were collected for assessment of alanine and aspartate aminotransferases as well as gamma glutamyl transferases. Liver samples were taken for detection of tumor necrosis factor-α (TNF-α) and malondialdhyde (MDA) levels as well as superoxide dismutase (SOD) activity. The mRNA expression of toll-like receptor-4 (TLR4), myeloid differentiation factor 88 (MyD88), high mobility group box 1 (HMGB1), and peroxisome proliferator-activated receptor-γ (PPAR-γ) were assessed in the liver. Hematoxylin& eosin stained liver sections were examined. Expressions of nuclear factor kappa B (NF-κB) and autophagy markers (LC3 II, P62) were assessed in immunostained sections. Results: Liver I/R increased serum transferases and tissue MDA, TNF-α, TLR4, HMGB-1, MyD88, NF-κB, LC3 II, P62 levels with hydropic degeneration and cellular infiltration in the liver while decreased SOD activity and PPAR-γ mRNA expression. These alterations were attenuated by paeonol pretreatment. Conclusions: Paeonol pretreatment alleviated liver I/R injury through inhibition of autophagy and TLR4/MyD88/NF-κB pathways.