Recently, gut flora has been linked to the onset of obesity and has been shown to influence the host's metabolism. Acupuncture is a well-known agent used for the treatment of numerous diseases such as obesity. This study aimed to explore the impacts of electroacupuncture treatment on gut microbiota composition and function in obese mice. Pyrosequencing of 16S rRNA genes and Metagenomic analysis of the fecal microbiota were used for this purpose. The basic parameters of body weight, Lee's index, serum lipid and epididymal adipose weight were ameliorated significantly after introducing an electroacupuncture intervention. Acidobacteria, Cyanobacteria and Basidiomycota (Normal group) and Fusobacteria, Firmicutes and Spirochmycetes (Model group) were remarkably affluent at the phylum level. Bacteroides sp. CAG: 927 and Prevotella sp. CAG: 1031 (Normal group), Lachnospiraceae bacterium and Helicobacter rodentium (Model group) at the species level were distinctly enriched. The structures and functions of the intestinal flora were significantly different between healthy and obese mice, and animals in the acupuncture group gradually tended towards healthy controls. Moreover, electroacupuncture altered the bacterial diversity and metabolic genes to establish new balance, observed the obvious change from 7[Formula: see text]d and stabilized gradually through 21[Formula: see text]d. These findings suggested gut flora could be a novel target of electroacupuncture treatment against obesity.