Gut microbiota play a key role in the regulation of obesity and associated metabolic disorders. To study the relationship between them, antibiotics have been widely used to generate pseudo-germ-free rodents as control models. However, it is not clear whether antibiotics impact an animal's metabolic phenotype. Therefore, the effect of antibiotics-induced gut microbial perturbations on metabolic phenotypes in high-fat diet (HFD) fed mice was investigated. The results showed that antibiotics perturbed gut microbial composition and structure. Community diversity and richness were reduced, and the phyla Firmicutes/Bacteroidetes (F/B) ratio was decreased by antibiotics. Visualization of Unifrac distance data using principal component analysis (PCA) and unweighted pair-group method with arithmetic mean (UPGAM) demonstrated that fecal samples of HFD-fed mice separated from those of chow diet (CD) fed mice. Fecal samples from antibiotics-treated and non-treated mice were clustered into two different microbial populations. Moreover, antibiotics suppressed HFD-induced metabolic features, including body weight gain (BWG), liver weight (LW), epididymal fat weight (EFW), and serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), fasting blood glucose (FBG), and insulin (INS) significantly (P<0.05). Lachnospiraceae, Ruminiclostridium and Helicobacter, biomarkers of mouse gut microbiota before treatment by antibiotics, were positively correlated with obesity phenotypes significantly (P<0.05) and were decreased by (92.95 +/- 5.09) %, (97.73 +/- 2.09) % and (99.48 +/- 0.21) % respectively after 30days of treatment by antibiotics. However, Bacteroidia were enriched in HFD-fed antibiotics-treated mice and were negatively correlated with obesity phenotypes significantly (P<0.05). We suggested that the antibiotics-induced depletion of Lachnospiraceae, Ruminiclostridium, and Helicobacter, and the decrease in F/B ratio in gut microbiota played a role in the prevention of HFD-induced obesity in mice.