Sirt1 is an NAD+-dependent deacetylase that plays a role in cellular processes such as transcriptional regulation, stress response, longevity and apoptosis. Sirt1 deacetylates histone proteins and certain transcription factors such as p53, CTIP2 (chicken ovalbumin upstream promoter-transcription factor-interacting protein 2), FOXO (forkhead box O) and NF-kappaB (nuclear factor kappaB). To identify potential Sirt1-interacting factors, we performed a yeast two-hybrid screen. The screen identified TLE1 (transducin-like enhancer of split-1) as a possible Sirt1-interacting factor, which was then confirmed by co-immunoprecipitation. TLE1 is a non-DNA binding co-repressor for several transcriptional factors including NF-kappaB. We have demonstrated using co-transfection assays that Sirt1 and TLE1 repress NF-kappaB activity. The catalytic mutant of Sirt1, Sirt1-H363Y, and the N-terminal Sirt1 fragment (amino acids 1-270) also show similar repression activity, suggesting that the deacetylase activity of Sirt1 may not be critical for its effect on NF-kappaB activity. Furthermore, analysis in Sirt1-null MEFs (murine embryonic fibroblasts) and HeLa cells stably expressing siRNA (small interfering RNA) specific to Sirt1 or TLE1 demonstrate that both Sirt1 and TLE1 are required for negative regulation of NF-kappaB activity. Taken together, these results suggest that the interaction between Sirt1 and TLE1 is important for mediating repression of NF-kappaB activity.