Natural products widely used due to various biologically activity such as anti-inflammatory, antioxidant, and anti-obesity. In this study, we were planned to determine the antioxidative and anti-obesity effects of Polygonum cuspidatum 50% ethanol extract (PEE). The antioxidative activity of PEE was calculated using the radical scavenging activity as well as total phenolic content, and reducing power. The anti-obesity effect of PEE was investigated using the 3T3-L1 adipocytes. The antioxidative activity of PEE was progressively increased in various concentration, mainly due to the presence of phenolic compounds. PEE also alleviated lipid accumulation on 3T3-L1 adipocytes and downregulated the mRNA and protein production of adipogenesis-related (SREBP-1c, PPARγ, C/EBPα) and lipogenesis-related (aP2, FAS, ACC) markers. Furthermore, we found that the inhibitory effect on lipid accumulation via PEE was caused by the alleviation of NF-κB, p38 MAPK, ERK1/2, and JNK at the protein level. Taken together, our results imply that PEE is a potential antioxidant that can prevent obesity-associated disorders.