Background and Purpose: Polyphenols of Polygoni Multiflori Caulis (PMC) have been widely consumed as food supplements for lowing lipid, while their anti-obesity effects were poorly understood. Experimental Approach: Several polyphenol-rich extracts including 75% ethanol (ETOH), chloroform (CHL), ethyl acetate (EA), n-butanol (n-Bu) and water (W) fractions, composed of emodin, physcion, apigenin or 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside at different ratios, were isolated from PMC. Their effects on lipase activity and 3T3-L1 preadipocytes differentiation were comparatively investigated. Their effects on obesity-related disorders were comparatively investigated using various models both in vitro and in vivo. Key Results: EA and n-Bu extracts with greater anti-obesity potential were screened for further studies to reveal the underlying mechanisms. EA and n-Bu extracts treatment significantly modulated the antioxidant response and lipid accumulation in Caenorhabditis elegans. High fat diet (HFD)-induced changes of weight gain, liver steatosis and epididymal adipose tissues hypertrophy in mice were also significantly reversed by EA and n-Bu extracts supplementation. Moreover, EA and n-Bu extracts intervention increased the microbial diversity, reshaped the microbiota structure and enhanced the relative abundance of Bifidobacterium. N-Bu extracts supplementation significantly decreased the ratio of Firmicutes/Bacteroidetes and the abundance of Lactobacillus, while EA extracts treatment markedly reversed HFD-induced increase of Erysipelotrichaceae abundance. Conclusions and Implications: Dietary supplementation of polyphenols from PMC exhibited promising anti-obesity effects and the chemical difference could partially explain their bioactivity discrepancy.