ObjectiveThis study aimed at establishing bacterial flagellin‐recognizing toll‐like receptor 5 (TLR5) as a novel link between gut microbiota composition, adipose tissue inflammation, and obesity.MethodsAn adipose tissue microarray database was used to compare women having the highest (n = 4, H‐TLR) and lowest (n = 4, L‐TLR) expression levels of TLR5‐signaling pathway genes. Gut microbiota composition was profiled using flow cytometry and FISH. Standard laboratory techniques were used to determine anthropometric and clinical variables. In vivo results were verified using cultured human adipocytes.ResultsThe H‐TLR group had higher flagellated Clostridium cluster XIV abundance and Firmicutes‐to‐Bacteroides ratio. H‐TLR subjects had obese phenotype characterized by greater waist circumference, fat %, and blood pressure (P < 0.05 for all). They also had higher leptin and lower adiponectin levels (P < 0.05 for both). Six hundred and sixty‐eight metabolism‐ and inflammation‐related adipose tissue genes were differentially expressed between the groups. In vitro studies confirmed that flagellin activated TLR5 inflammatory pathways, decreased insulin signaling, and increased glycerol secretion.ConclusionsThe in vivo findings suggest that flagellated Clostridium cluster XIV bacteria contribute to the development of obesity through distorted adipose tissue metabolism and inflammation. The in vitro studies in adipocytes show that the underlying mechanisms of the human findings may be due to flagellin‐activated TLR5 signaling.