Objective：To study the relationship between mitochondrial augmenter of liver regeneration（ALR）and mitochondrial dysfunction in cell model of hepatocyte lipotoxicity. Methods：The 10% fatty acid-free bull serum albumin treated L-O2 cells were used as the control group（BSA group），and L-O2 cells treated with 0.2 mmol/L palmitic acid（PA）as a cell model of hepatocyte lipotoxicity（PA group）. Meanwhile，the ALR overexpression group（ALR-OE group）and the empty vector transfected control cell group（Vector group）were constructed and treated with BSA or PA，respectively. cell viability，lactate dehydrogenase（LDH）release，mitochondrial membrane potential changes，apoptosis and related protein expression levels were detected subsequently. Results：Compared with the BSA group，the intracellular lipid droplet increased，the cell viability decreased by about 50%，and the LDH release increased by 13 times，the expression of ALR in the mitochondria was significantly decreased in the PA group. There were no significant differences between the Vector group and the ALR-OE group under BSA treatment conditions. However，the cell viability increased by about 16%，LDH release is reduced by about 40%，mitochondrial membrane potential is increased by about 18%，cytochrome C release is reduced，and apoptosis decreases in the ALR-OE group compared with the Vector group under PA stimulation. Conclusion：ALR is involved in the onset of hepatocellular lipotoxicity，and targeting the regulation of mitochondrial ALR can inhibit the onset of lipotoxicity to a certain extent.