SIRT1 is a NAD+ dependent protein deacetylase. Nicotinamide is a reaction product of the SIRT1 catalyzed deacetylation and also a noncompetitive inhibitor of SIRT1. Here we built the complex structures of “SIRT1/NAD+/p53 acetyl peptide” and “SIRT1/ADPR/nicotinamide” by protein homology modeling and molecular docking. According to the docking results, S265 and N346 of SIRT1 are involved in the interactions with NAD+, while S275 is at the entrance of the NAD+ binding pocket. Consistently, our enzyme kinetics studies proved that mutations of S265, S275 or N346 either reduced or destroyed the interaction of NAD+ with SIRT1. In addition, the carboxamide group of nicotinamide forms hydrogen bonds with I347 and D348, while the pyridine ring is buried in a hydrophobic environment surrounded by A262, I270, F273, I316 and I347. Interestingly, mutation of I316 to alanine increased the affinity of nicotinamide and dramatically improved its inhibitory activity, indicating that nicotinamide binds in the C pocket of SIRT1.