Resveratrol has been shown to possess multiple pharmacological activities including anti-tumor, anti-inflammation and immunomodulation, and participates in the regulation of the NF-κB signaling pathway. However, the mechanism of the NF-κB signaling pathway inhibited by resveratrol remains obscure. In this study, we first examined the effect of resveratrol on endogenous and TNF-α-induced NF-κB activation, and found that resveratrol suppressed NF-κB activation in a dose dependent manner. Resveratrol reduced the transcriptional activity of p65, but neither affected the DNA-binding activity of NF-κB nor blocked the nuclear translocation of p65. Moreover, resveratrol had no effect on the expression level of IκBα protein and inhibited IκBα degradation. Further investigation revealed that resveratrol blocked the ubiquitination of NEMO and inhibited IκB kinaseβ-mediated NF-κB activation. These results demonstrated that resveratrol effectively suppressed NF-κB signaling through inhibiting the activities of NF-κB and IκB kinase. Therefore, resveratrol may provide a novel approach to treating inflammation-associated diseases and cancer.