Thiolate-protected Ag nanoparticles (NPs) exhibit interesting physical and chemical properties which may lead to various sensing, diagnostic, and therapeutic applications. Further, understanding structure–property relationships of Ag NPs is of great interest to optimize their application. Herein, we used TEM, UV–vis, and a series of synchrotron X-ray spectroscopy techniques to probe the local structure and chemical bonding properties of thiolate-stabilized Ag NPs. Compared with other Ag nanostructures prepared under slightly modified conditions, the Ag NPs were found to have pronounced structural changes, which led to immensely different optical properties. Notably, the NPs were also found to have similar surface structure to recently elucidated Ag nanoclusters prepared with different thiolates. These findings suggest that the NP structure and optical properties can be sensitively tailored by controlling the synthetic conditions. The multi-element, multi-core excitation approach (i.e., Ag K-, Ag L 3 -, and S K-edges) employed in the X-ray absorption spectroscopy measurements was also demonstrated as an effective tool to uncover the NP structure from both the metal core and the ligand shell perspectives.