Single-atom catalysts have become the forefront and hotspot in catalysis research due to their unique structural characteristics such as extreme atomic utilization efficiency and uniformity of reaction centers. To break through the bottleneck on their intrinsic activity enhancement, arduous efforts have been made to regulate the metal single-atomic sites. Herein, with a special interest in interatomic interfaces and interactions, our review focuses on the atomic-level regulation strategies of single-atom catalysts, including nonmetal heteroatom doping and polymetallic active site construction, referring to the introduction of nonmetal or metal atoms, respectively, into primitive metal single-atomic sites. We summarize the progress of these two strategies in optimizing single-atom active sites from the perspectives of synthesis, application, and mechanism. We start from a systematic overview of the atomic-level regulation methods and corresponding fabrication routes, followed by a comprehensive summary of the current advances in some typical electrocatalytic reactions. Then we expound on the synergistic reaction mechanism and the intrinsic structure-activity relationship after regulation. In the final part, we discuss the challenges and opportunities of these two atomic-level regulation strategies for further development. Our review of the atomic-level regulation of single-atom catalysts gives significant insight into the rational design of atom-efficient catalysts and adds depth and breadth to the fundamental understanding of atomically precise catalysis by elucidating its intrinsic mechanisms.