5-Hydroxymethylfurfural(5-HMF), a water-soluble compound extracted from wine-processed Fructus corni, is a novel hepatic protectant for treating acute liver injury. The present study was designed to investigate the protective effect of 5-HMF in human L02 hepatocytes injured by D-galactosamine(Gal N) and tumor necrosis factor-α(TNF-α) in vitro and to explore the underlying mechanisms of action. Our results showed that 5-HMF caused significant increase in the viability of L02 cells injured by Gal N/TNF-α, in accordance with a dose-dependent decrease in apoptotic cell death confirmed by morphological and flow cytometric analyses. Based on immunofluorescence and Western blot assays, we found that Gal N/TNF-α induced ER stress in the cells, as indicated by the disturbance of intracellular Ca2+ concentration, the activation of protein kinase RNA(PKR)-like ER kinase(PERK), phosphorylation of eukaryotic initiation factor 2 alpha(e IF2α), and expression of ATF4 and CHOP proteins, which was reversed by 5-HMF pre-treatment in a dose-dependent manner. The anti-apoptotic effect of 5-HMF was further evidenced by balancing the expression of Bcl-2 family members. In addition, the knockdown of PERK suppressed the expression of phospho-PERK, phospho-e IF2α, ATF4, and CHOP, resulting in a significant decrease in cell apoptosis after the treatment with Gal N/TNF-α. 5-HMF could enhance the effects of PERK knockdown, protecting the cells against the Gal N/TNF-α insult. In conclusion, these findings demonstrate that 5-HMF can effectively protect Gal N/TNF-α-injured L02 hepatocytes against ER stress-induced apoptosis through the regulation of the PERKe IF2α signaling pathway, suggesting that it is a possible candidate for liver disease therapy. 5-Hydroxymethylfurfural (5-HMF), a water-soluble compound extracted from wine-processed Fructus corni, is a novel hepatic protectant for treating acute liver injury. The present study was designed to investigate the protective effect of 5-HMF in human L02 HMG caused by D-galactosamine (Gal N) and tumor necrosis factor-alpha (TNF-alpha) in vitro and to explore the underlying mechanisms of action. Our results showed that 5-HMF caused significant increase in the viability of L02 cells injured by Gal N / TNF-α, in accordance with a dose-dependent decrease in apoptotic cell death confirmed by morphological and flow cytometric analyzes. Based on immunofluorescence and Western blot assays, we found that Gal N / TNF-α-induced ER stress in the cells , as indicated by the disturbance of intracellular Ca2 concentration, the activation of protein kinase RNA (PKR) -like ER kinase (PERK), phosphorylation of eukaryotic initiation factor 2 alpha (e IF2α), and expression of ATF4 and CHOP proteins, which was The anti-apoptotic effect of 5-HMF was further evidenced by balancing the expression of Bcl-2 family members. In addition, the knockdown of PERK suppressed the expression of phospho -PERK, phospho-e IF2α, ATF4, and CHOP, resulting in a significant decrease in cell apoptosis after the treatment with Gal N / TNF-α. 5-HMF could enhance the effects of PERK knockdown, protecting the cells against the Gal N / TNF-α insult. In conclusion, these findings demonstrate that 5-HMF can effectively protect Gal N / TNF-α-injured L02 hepatocytes against ER stress-induced apoptosis through the regulation of the PERKe IF2α signaling pathway, suggesting that it is a possible candidate for liver disease therapy.