Recent advances in Au–thiolate nanocluster synthesis have allowed the total structural determination of several nanoclusters by X-ray crystallography. The high-precision structural information of these nanoclusters enables atomic site-specific analysis of local structure and electronic character. In this work, a site-specific comparative study of Au102(SR)44 and Au25(SR)18 was conducted to elucidate the size and site effects on the local environment and electronic character of their common surface structural unit, the –SR–Au–SR–Au–SR– “double-staple” motif. Simulation of the pseudo-radial distribution function from extended X-ray absorption fine structure (EXAFS) shows a significant difference in their local environments despite their identical geometric shape. Local density of states (l-DOS) calculations consistently reveal the difference in their electronic characters for gold d-electron density and d-DOS position. These differences are then related to the unique aurophilic interactions and size- or site-dependent electronic character of Au atoms in the double-staple motif. The differing local structure and electronic behaviour of the “double-staple” motif in Au102(SR)44 and Au25(SR)18 highlight the significance of both size and site effects on the surface structure and electronic property of Au–thiolate nanoclusters. The theoretical results may also be useful in the interpretation of future experimental XAFS and X-ray photoelectron spectroscopy (XPS) data of these nanoclusters.