Fucosylated chondroitin sulfate from Pearsonothuria graeffei (FCS-Pg), a natural macromolecular polysaccharide, has been proven to prevent obesity, but its underlying molecular mechanism is still unclear. C57BL/6 J mice fed on high fat diet (HFD) were administered FCS-Pg lasting for ten weeks. The results demonstrated that FCS-Pg supplementation reduced body weight with dosage manner compared with HFD group. The expressions of intestinal lipid synthesis related proteins such as cluster of differentiation 36 (CD36) in FCS-Pg group were lower than those in the HFD group. Compared with the normal group, HFD caused gut microbiota disorder in the colon. FCS-Pg supplementation at high dosage restored the gut microbiota composition with higher abundance of Alistipes and Bacteroidetes and lower abundance of Colidextribacter, Bilophila and Firmicutes compared with HFD group. Moreover, FCS-Pg decreased the expression of proteins involved in triglyceride synthesis such as glycerol 3-phosphate dehydrogenase 1 (GPD1) and increased the expression of proteins involved in lipolysis and thermogenesis such as adipose triglyceride lipase (ATGL) and uncoupling protein 1 (UCP1) in the white adipose tissue (WAT) compared with HFD group. In conclusion, our study suggested that FCS-Pg significantly prevented obesity and improved WAT function in HFD-fed mice by regulating intestinal lipid metabolism and microflora composition.