Inhibitory effect of fucosylated chondroitin sulfate from the sea cucumber Acaudina molpadioides on adipogenesis is dependent on Wnt/β-catenin pathway. The fucosylated chondroitin sulfate was isolated from the sea cucumber Acaudina molpadioides (Am-CHS) and its effect on the adipogenesis was investigated in vitro. Results showed that Am-CHS exhibited a marked anti-adipogenic effect. The TG contents of cells treated with Am-CHS from the early stage, mid stage and later stage were decreased by 31.76%, 19.76% and 13.85%, respectively. Wnt/β-catenin pathway acts as a negative regulator of adipogenesis. It is showed that Am-CHS enhanced the expression of the Wnt/β-catenin related factors, namely Wnt10b, β-catenin, Fz and LRP, causing more cytoplasmic β-catenin translocated into the nuclear to inhibit the expression of PPARγ and C/EBPα. SREBP-1c promotes the adipocyte differentiation by enhancing the activity of PPARγ. Reverse transcriptase-polymerase chain reaction assay and western blotting results revealed that the expressions of SREBP-1c and its transcriptional product FAS were inhibited by Am-CHS. Further study showed that Am-CHS also suppressed the PPARγ and C/EBPα expression. These results indicate that the anti-adipogenic activity of Am-CHS may exhibited by activating the Wnt/β-catenin pathway and down-regulating the SREBP-1c expression. To further verify this conclusion, in vivo experiments were performed. Results revealed that Am-CHS reduced the body adipose rate compared to high-fat-high sucrose diet (HFSD)-fed mice. Western blotting results showed that Am-CHS increased the expression of β-catenin, a negative regulator in adipogenesis, and decreased that of SREBP-1c, PPARγ and C/EBPα, the positive regulators of adipogenesis in the adipose tissue compared to the HFSD-fed group. These results further confirmed the anti-adipogenic activity of Am-CHS.