In this study, we investigated the effect of 7-O-galloyl-d-sedoheptulose (GS) on adipocyte differentiation in 3T3-L1 cells. GS significantly suppressed 3T3-L1 differentiation. The activation of adenosine monophosphate-activated protein kinase (AMPK) was demonstrated by measuring phosphorylation. In addition, GS down-regulated the adipogenesis-related protein expression of the sterol regulatory element-binding protein 1c. GS significantly reduced the protein expression of the peroxisome proliferator-activated receptor γ and of the CCAAT/enhancer-binding protein α, two key transcription factors in adipogenesis. These findings indicate that GS exerts anti-adipogenic activity via AMPK activation and may act to prevent obesity.