Toll-like receptor 5 (TLR5) recognizes the bacterial product, flagellin. Its activation leads to cytokine production that is important for host defense against invading pathogens. In spite of the pro-inflammatory nature of TLR5, deficiency of TLR5 exacerbated metabolic dysfunction in diet-induced obese mouse model (Gewirtz et al. Science, 328: 228-231, 2010). In this study, we found that TLR5 was expressed in both preadipocytes and adipocytes, and tried to characterize the functions of TLR5 in these metabolic cells Six week old wild type (WT) and TLR5-knockout (T5KO) mice were fed a high fat diet for five weeks, andthey both showed a similar body weight gain and daily food intake, but NMR spectroscopy revealed a decreased whole-body fat mass in T5KO mice. Particularly, gonadal adipose tissues in T5KO mice were significantly smaller compared with those in WT, whereas the adipose inflammatory status was unaltered. The in vitro experiments showed that TLR5-deficient stromal vascular cells isolated from visceral fat depots had reduced adipogenic capacity. In addition, elevated triglyceride accumulation was observed in livers from T5KO mice. iIt is possible that TLR5 pathway participates in adipogenesis to mediate adipose tissue expansion and prevents ectopic storage of lipids in liver after high fat diet feeding. It is noteworthy to further explore the possible direct functions of TLR5 in metabolic cells.