To investigate the effect and potential mechanism of chitooligosaccharides on reducing liver lipid accumulation in vitro, sodium oleate was used to induce HepG2 cells to establish non-alcoholic fatty liver cell model, the lipid profiles including total lipid accumulation, total triglyceride, total cholesterol, high/low density lipoprotein cholesterol and free fatty acids contents were analyzed by oil red O staining and corresponding commercial kits, respectively. The expression of genes and proteins related to intracellular lipid metabolism were detected by real-time PCR and western blot. The results indicated that 4.0mg/mL chitooligosaccharides had a significant inhibitory effect on liver lipid accumulation and reduced the mRNA and protein expression levels of diacylglycerol acyltransferase 2 (DGAT2), liver X receptor alpha (LXRα), peroxisomes proliferator-activated receptor-γ (PPARγ), pregnenolone X receptor (PXR) and differentiation cluster 36 (CD36). These results suggested that chitooligosaccharides could reduce the accumulation of lipids by regulating fatty acid absorption and triglyceride synthesis pathways. This study aims to provid theoretical support for using chitooligosaccharides as dietary supplements in lipid-lowering and liver protection.