Obesity has become a serious public health threat worldwide and recent evidence demonstrates that gut microbiota plays an important role in obesity. To the best of our knowledge, no research has been presented demonstrating the effects of a chlorophyll-rich spinach extract supplementation on obesity and associated changes in gut microflora of mice. The present study found that a chlorophyll-rich spinach extract supplementation effectively alleviated body weight gain and low-grade inflammation, and improved the glucose tolerance in HFD-fed mice (p < 0.05). High-throughput sequencing of the 16S rRNA showed the chlorophyll-rich spinach extract supplementation reversed HFD-induced gut microbiota dysbiosis, which was likely associated with the increased abundance of norank_f_Bacteroidales_S24-7_group and Akkermansia, and the decreased abundance of Lactobacillus and Lactococcus in mice. Taken together, this study suggested that the prevention of obesity by the chlorophyll-rich spinach extract supplementation was at least partially mediated by structural modulation of gut microbiota.