Nonalcoholic fatty liver disease (NAFLD) is a global epidemic, and there is no standard and efficient therapy for it. Chitosan oligosaccharide (COS) is widely known to have various biological effects, and in this study we aimed to evaluate the liver-protective effect in diet-induced obese mice for an enzymatically digested product of COS called COS23 which is mainly composed of dimers and trimers. An integrated analysis of the lipidome and gut microbiome were performed to assess the effects of COS23 on lipids in plasma and the liver as well as on intestinal microbiota. Our results revealed that COS23 obviously attenuated hepatic steatosis and ameliorated liver injury in diet-induced obese mice. The hepatic toxic lipids-especially triglycerides (TGs) and free fatty acids (FFAs)-were decreased dramatically after COS23 treatment. COS23 regulated lipid-related pathways, especially inhibiting the expressions of FFA-synthesis-related genes and inflammation-related genes. Furthermore, COS23 could alter lipid profiles in plasma. More importantly, COS23 also decreased the abundance of Mucispirillum and increased the abundance of Coprococcus in gut microbiota and protected the intestinal barrier by up-regulating the expression of tight-junction-related genes. In conclusion, COS23, an enzymatically digested product of COS, might serve as a promising candidate in the clinical treatment of NAFLD.