Objective To explore the protective effect of cannabidiol (CBD) on palmitic acid (PA)-induced hepatocytes injury and to investigate the underlying mechanism associated with autophagic flux. Methods Primary cultured rat hepatocytes were treated with 1 or 5 μmol/L CBD for 24 h and Western blotting was performed to detect autophagy-related protein microtubule-associated protein 1 light chain 3 (LC3) and p62 expressions. Then the hepatocytes were divided into 4 groups and received different interventions, including PA group (the hepatocytes were stimulated with 800 μmol/L PA), PA+CBD group (the hepatocytes were co-stimulated with 800 μmol/L PA and 5 μmol/L CBD), PA+CBD+CQ group (the hepatocytes were co-stimulated with 800 μmol/L PA, 5 μmol/L CBD and 50 nmol/L autophagy inhibitor chloroquine[CQ]) and negative control group (an equal volume of 0.03% DMSO was added to the culture medium); the hepatocytes in all groups were treated for 24 h. We used Western blotting to detect LC3 and p62 proteins expressions, flow cytometry to determine apoptosis, qPCR assay to detect the mRNA expressions of CCAAT/enhancer-binding protein homologous protein (CHOP), glucose-regulated protein 78 (GRP78) and X-box-binding protein 1 (XBP-1), and the Rh123 and lucigenin fluorescent probes to detect the mitochondrial membrane potential and reactive oxygen species (ROS) content, respectively. Results The ratio of LC3-Ⅱ to LC3-Ⅰand p62 protein expression had no change in the cultured rat hepatocytes treated with 1 or 5 μmol/L CBD. Compared with the negative control group, the ratio of LC-Ⅱ to LC3-Ⅰand p62 protein expression were significantly increased (P<0.05), the apoptosis was significantly risen (P<0.05), the mRNA expressions of CHOP, GRP78 and XBP-1 were significantly increased (P<0.05), mitochondrial membrane potential was significantly decreased (P<0.05), and the ROS content in mitochondrial was significantly increased (P<0.05) in the PA group. Compared with PA group, the hepatocytes in the PA+CBD group showed an improved autophagic flux, increased apoptosis, reduced endoplasmic reticulum stress and mitochondrial dysfunction (all P<0.05). The protective effect of CBD on PA-induced hepatocytes injury was significantly inhibited by co-incubation with CQ (P<0.05). Conclusion CBD can attenuate PA-induced hepatocytes injury through promoting autophagic flux, reducing hepatocyte apoptosis, and improving endoplasmic reticulum stress and mitochondrial dysfunction.