Lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria serve as endotoxin to exert potent immune responses. However, the effect of LPS on adipogenesis has not been elucidated. The present study was designed to examine the effect of LPS on adipogenesis in 3T3-L1 preadipocytes and possible mechanism(s) of action involved. Our results revealed that LPS challenge significantly suppressed adipogenesis in 3T3-L1 preadipocytes mainly through downregulated expression of the late adipogenic markers PPARγ and aP2 as well as AMP-activated protein kinase (AMPK) expression and activity. As an inflammatory factor, LPS was found to lead to an overt reduction in IκBα levels compared with the time-matched controls, consolidating its pro-inflammatory property in 3T3-L1 preadipocytes. Our data also revealed that LPS retarded adipogenesis, the effect of which was partially reversed by the selective inhibitor of IKKβ. IκBα was found to be involved in the anti-adipogenic effect of LPS. In conclusion, LPS is capable of inhibiting adipogenesis in 3T3-L1 adipocytes possibly through activation of NF-κB and inhibition of AMPK. With the activation of NF-κB pathway and inhibition of AMPK, LPS suppresses C/EBP α DNA-binding activity and the expression of late adipogenic markers PPARγ and aP2.