SUMMARY The NAD+-dependent protein deacetylase SIRT1 regulates energy metabolism, responses to stress, and aging by deacetylating many different proteins, including histones and transcription factors. The mechanisms controlling SIRT1 enzymatic activity are complex and incompletely characterized, yet essential for understanding how to develop therapeutics that target SIRT1. Here we demonstrate that the N-terminal domain of SIRT1 (NTERM) can trans-activate deacetylation activity by physically interacting with endogenous SIRT1 and promoting its association with the deacetylation substrate NF-κB p65. Two motifs within the NTERM domain contribute to activation of SIRT1-dependent activities, and expression of one of these motifs in mice is sufficient to lower fasting glucose levels and improve glucose tolerance in a manner similar to overexpression of SIRT1. Our results provide new insights into the regulation of SIRT1 activity and a rationale for pharmacological control of SIRT1-dependent activities.