In this study, we investigated the catalytic properties of N,N-dimethylformamide (DMF)-stabilized gold nanoclusters (AuNCs) in the reduction of 4-nitrophenol (PNP) to 4-aminophenol by NaBH4, a well known model reaction to be catalyzed by metal surfaces. The DMF-stabilized AuNCs were prepared in DMF by a surfactant-free method. The DMF-stabilized AuNCs showed high catalytic activity even when used in small quantities (∼10−7 g). The pseudo-first-order rate constant (kapp) and activation energy were estimated to be 3 × 10−3 s−1 and 31 kJ mol−1, respectively, with 1.0 μM of the gold catalyst at 298 K. The catalytic activity of the DMF-stabilized AuNCs was strongly influenced by the layer of adsorbed DMF on the Au NCs. This layer of adsorbed DMF prohibited the reactants from penetrating to the surface of the AuNCs via the diffusion at the beginning of the reaction, resulting in an induction time (t0) before PNP reduction began. Restructuring of the DMF layer (essentially a form of activation) was the key to achieving high catalytic activity. In addition, atomically monodisperse Au25(SG)18NCs (SG: glutathione) showed higher catalytic activity in the PNP reduction (kapp = 8 × 10−3 s−1) even with a low catalyst concentration (1.0 μM), and there was no induction time (t0) in spite of the strongly binding ligand glutathione. This suggested that the catalytically active surface sites of the Au25(SG)18NCs were not sterically hindered, possibly because of the unique core–shell-like structure of the NCs. Retaining these open sites on AuNCs may be the key to making the NCs effective catalysts.