As an interdisciplinary product, water-soluble gold nanoclusters (AuNCs) stabilized by ligands containing carboxyl (–COOH) group have garnered significant attention from synthetic chemists and biologists due to their immense potential for biomedical applications. However, revealing the crystallographic structures of –COOH-functionalized AuNCs remains a bottleneck. Herein, we successfully applied the salting-out method to obtain a series of high-quality single crystals of –COOH-functionalized Au25 nanoclusters and revealed their crystallographic structures. Particularly, K3Au25(2-Hmna)9(mna)6]− (Au25a) protected by 2-mercaptonicotinic acid features an unprecedented tetrameric Au4(SRS)3(SRS,N)2 staple motifs surrounding the icosahedral Au13 kernel, breaking the traditional perception on the structure of Au25(SR)18. Au25a exhibits a distinct near-infrared emission at 970 nm with long lifetime of 8690 ns, which have been studied by transient absorption spectroscopy and time-dependent density functional theory. This work compensates for the research gap in the experimental structure of –COOH-functionalized AuNCs and opens up a new avenue to explore their structure–property correlations.