Acupuncture has been used to treat numerous diseases such as obesity in China for thousands of years. Several mechanisms of acupuncture on obesity have been surveyed based on metabolomics, but the effects of acupuncture on the alterations in the gut flora are still unclear. In this study, an integrated approach based on 16S rRNA gene sequencing combined with high-performance liquid chromatography-mass spectrometry (HPLC-MS) metabolic profiling was conducted to investigate the effects of acupuncture on high-fat-diet-induced obesity through the regulation of the relative abundances of gut microbiota and their relationships with biomarker candidates. A total of 10 significantly altered bacterial genera and 11 metabolites were recognized, which recovered to normal levels after electroacupuncture treatment. The relative abundances of the bacterial families ,,,, ,,,, andwere remarkedly changed among the three groups. Potential biomarkers, including (0:0/16:0) ([Formula: see text]1),(0:0/18:0) ([Formula: see text]2),([Formula: see text]3),(16:0) ([Formula: see text]4), 3[Formula: see text],6[Formula: see text],7[Formula: see text]--5[Formula: see text]-([Formula: see text]5), 5-([Formula: see text]6),(18:0/0:0) ([Formula: see text]7), 1--5--6--5,6-([Formula: see text]8),([Formula: see text]9),[Formula: see text]-([Formula: see text]10) and([Formula: see text]11), were involved in several metabolic pathways, such as the glycerophospholipid metabolism and primary bile acid biosynthesis. Interestingly, there was a strong correlation between the perturbed gut flora in and and the altered intestinal metabolite of 3[Formula: see text],6[Formula: see text],7[Formula: see text]--5[Formula: see text]- and and [Formula: see text]-. This finding suggested that the effects of electroacupuncture might change the proportions of and by regulating the constituents of the functional metabolite of 3[Formula: see text],6[Formula: see text],7[Formula: see text]--5[Formula: see text]- and and [Formula: see text]-. These results indicated that the effects of electroacupuncture focused on custom metabolic pathways as well as depend on the changes in the gut microbiota in obesity. These findings suggest that the 16S rRNA gene sequencing and HPLC-MS-based metabolomics approach can be applied to comprehensively assess the effects of traditional Chinese medicines.