Previous studies have found that chitosan oligosaccharide (COST) can alleviate the clinical symptoms in non-alcoholic fatty liver disease (NAFLD) patients. We intend to intervene with different concentrations of COST in mice with NAFLD induced by a high fat diet. The basic effect of COST on NAFLD model mice was observed using physiological and biochemical indexes. 16S rRNA sequencing technology was used to analyze the gut microbiota and further analyze the content of short-chain fatty acids (SCFAs). Western blot and RT-PCR were used to detect the effects of COST on the PI3K/AKT/mTOR signaling pathway in the livers of NAFLD mice. It was found that the COST-high-dose group could reduce the weight of NAFLD mice, improve dyslipidemia, and alleviate liver lesions, and COST has a therapeutic effect on NAFLD mice. 16S rRNA sequencing analysis showed that COST could increase the diversity of the gut microbiota in NAFLD mice. The downregulation of SCFAs in NAFLD mice was reversed. WB and RT-PCR results showed that the PI3K/AKT/mTOR signaling pathway was involved in the development of NAFLD mice. COST improved liver lipid metabolism in NAFLD mice by inhibiting liver DNL. COST could increase the expression of thermogenic protein and UCP1 and PGC-1α genes; the PI3K/AKT/mTOR signaling pathway is inhibited at the protein and gene levels. This study revealed that COST regulates the expression of related inflammatory factors caused by lipid toxicity through the gut microbiota and SCFAs, and improves the liver lipid metabolism of HFD-induced NAFLD mice, laying a foundation for the development of effective and low toxicity drugs for the treatment of NAFLD.