Xanthigen is a source of punicic acid and fucoxanthin derived from pomegranate seed and brown seaweed, respectively with recognized triacylglycerol-lowering effects in humans, yet the mechanism remains to be fully elucidated. The present study investigated the inhibitory effects of Xanthigen, fucoxanthin, and punicic acid (70% in pomegranate seed oil) on the differentiation of 3T3-L1 preadipocytes. Xanthigen potently and dose-dependently suppressed accumulation of lipid droplets in adipocytes compared to its individual components, fucoxanthin and pomegranate seed oil. Western blot analysis revealed that Xanthigen markedly down-regulated the protein levels of key adipogenesis transcription factors peroxisome proliferator-activated receptor (PPAR)γ, CCAAT/enhancer binding protein (C/EBP) β, and C/EBPδ as well as a key enzyme involved in adipogenesis, fatty acid synthase (FAS). Xanthigen up-regulated the NAD(+)-dependent histone deacetylases (SIRT1) and activated AMP-activated protein kinase (AMPK) signaling in differentiated 3T3-L1 adipocytes. In addition, Xanthigen may also stimulate insulin trigger signaling and result in Akt-dependent phosphorylation of forkhead/winged helix O (FoxO)1 and FoxO3a. These results indicate that Xanthigen suppresses adipocyte differentiation and lipid accumulation through multiple mechanisms and may have applications for the treatment of obesity.